JP7379803B2 - Multispecific chimeric receptors containing NKG2D domains and their uses - Google Patents

Description

[Cross reference to related applications]
This application claims the benefit of priority from International Patent Application No. PCT/CN2017/119397, filed on December 28, 2017, the contents of which are hereby incorporated by reference in their entirety. . Submission of sequence listing as ASCII text file

The contents of the following submission as an ASCII text file are hereby incorporated by reference in its entirety: Computer Readable Form (CRF) of the Sequence Listing (File Name: 761422000941SEQLISTING.txt, Date of Recording: 2018 December 28th, size: 85KB).

The present invention relates to chimeric receptors, multispecific chimeric receptors, dual chimeric receptor systems, engineered immune effector cells, and methods of use thereof.

Acute myeloid leukemia (AML) is characterized by an abnormal clonal expansion of bone marrow precursors that predominate in the bone marrow and blood, and this abnormal clonal expansion severely impairs normal hematopoiesis. AML is the most common type of acute leukemia, accounting for approximately 25% of all adult-onset leukemias in Western countries. AML has an incidence of 3-5 per 100,000 adults per year and the highest mortality rate of all leukemias (DiNardo and Cortes 2016). Over the past 40 years, the 5-year relative overall survival rate for AML patients has increased slightly, from an abysmal 6.3% between 1975 and 1980 to 23.9% between 2007 and 2012 (Mardiros et al. 2015).

The standard first-line treatment for AML is chemotherapy using cytarabine in combination with an anthracycline as induction therapy, followed by high-dose cytarabine and/or congeners for patients who achieve complete remission (CR) after induction therapy. Repeated cycles of stem cell transplantation (alloSCT) follow. Initial CR can be achieved in approximately 70% of young adult patients with current induction chemotherapy, but 43% of patients later relapse and 18% never achieve CR using first-line therapy ( Forman and Rowe 2013). alloSCT is the preferred treatment after second remission. The 5-year disease-free survival rate among patients undergoing alloSCT is 40-50%, indicating sensitivity to immune-based treatments for AML. However, patients with primary refractory disease or with an initial CR duration of less than 6 months benefit little from alloSCT treatment (Mardiros et al. 2015). Additionally, cytotoxic damage to patient organs from conventional salvage chemotherapy further reduces the likelihood of success for alloSCT. Therefore, there is a need for more effective and less invasive treatments for AML patients after relapse or failed induction.

T cells have the potential to attack and eliminate tumors, especially those with a high number of genetic mutations that cause neoantigens. However, the antitumor capacity of T cells is often actively inhibited by the immunosuppressive tumor microenvironment (TME) (McGranahan et al. 2016). Chimeric antigen receptor T cells (CAR-Ts) contain an extracellular antigen-binding domain that is directed toward antigens on tumor cells, a hinge region, and a T-cell receptor (TCR) for triggering T-cell activation upon antigen binding. ) by transducing a gene encoding a fusion protein containing an intracellular signaling domain. Unlike conventional T cells, which rely on native TCRs for tumor antigen recognition, CAR-T cells are redirected to unprocessed antigens and are thereby independent of major histocompatibility complex (MHC) antigen expression. to kill tumor cells. Therefore, CAR-T cells have the ability to overcome many of the limitations inherent in immunotherapy. After 20 years of preclinical research and clinical trials, the safety and feasibility of CAR-T-based therapy has been confirmed, and unprecedented clinical results have been obtained in hematological malignancies (Kochenderfer et al. .2015; Louis et al. 2011).

The disclosures of all publications, patents, patent applications, and published patent applications mentioned herein are hereby incorporated by reference in their entirety.

The present application provides multispecific chimeric receptors and dual chimeric receptor systems that target an NKG2D ligand and a second antigen, such as a tumor antigen, eg, CD123. Chimeric receptors targeting NKG2D ligands are also provided.

One aspect of the present application provides a chimeric receptor having (a) an extracellular domain comprising an NKG2D domain, (b) a transmembrane domain, and (c) an intracellular signaling domain. In some embodiments, the extracellular domain includes a first NKG2D domain and a second NKG2D domain.

One aspect of the present application provides a multispecific chimeric receptor having (a) an extracellular domain comprising an NKG2D domain and a second antigen binding domain, (b) a transmembrane domain, and (c) an intracellular signaling domain. Provide your body.

One aspect of the present application provides (a) an extracellular domain comprising a first NKG2D domain, a second NKG2D domain, and a second antigen-binding domain, (b) a transmembrane domain, and (c) an intracellular domain. A multispecific chimeric receptor is provided comprising a polypeptide chain having a signaling domain. In some embodiments, the extracellular domain includes, from N-terminus to C-terminus, a second antigen binding domain, a first NKG2D domain, and a second NKG2D domain. In some embodiments, the second antigen binding domain is fused to the first NKG2D domain via a peptide linker. In some embodiments, the peptide linker is about 50 amino acids or less in length. In some embodiments, the peptide linker comprises an amino acid sequence selected from SEQ ID NOs: 12-15.

One aspect of the present application provides a multispecific chimeric receptor comprising a first polypeptide chain and a second polypeptide chain, each polypeptide chain comprising (a) an NKG2D domain and a second antigen. (b) a transmembrane domain; and (c) an intracellular signaling domain. In some embodiments, each extracellular domain further comprises a dimerization motif. In some embodiments, the dimerization motif is transferred between the NKG2D domain and the second antigen binding domain. In some embodiments, the dimerization motif is a leucine zipper or a cysteine zipper. In some embodiments, the second antigen binding domain is fused to the NKG2D domain via a peptide linker. In some embodiments, the peptide linker is about 50 amino acids or less in length. In some embodiments, the peptide linker comprises an amino acid sequence selected from SEQ ID NOs: 12-15. In some embodiments, the NKG2D domain of the first polypeptide chain is cross-linked to the NKG2D domain of the second polypeptide chain by one or more disulfide bonds. In some embodiments, each of the first polypeptide chain and the second polypeptide chain, from N-terminus to C-terminus, comprises a second antigen binding domain, an NKG2D domain, a transmembrane domain, and an intracellular signaling domain. Contains domains.

In some embodiments according to any one of the multispecific chimeric receptors described above, the multispecific chimeric receptor is a bispecific chimeric receptor.

In some embodiments according to any one of the multispecific chimeric receptors described above, the second antigen binding domain is an antibody fragment. In some embodiments, the antibody fragment is CD19, CD20, CD22, CD33, CD38, BCMA, CS1, ROR1, GPC3, CD123, CD138, c-Met, EGFR, EGFRvIII, HER2, HER3, GD-2, NY - specifically binds to an antigen selected from the group consisting of ESO-1, MAGE A3, and glycolipid F77. In some embodiments, the second antigen binding domain is a ligand or a ligand binding domain. In some embodiments, the ligand or ligand binding domain is derived from a molecule selected from the group consisting of NKG2A, NKG2C, NKG2F, IL-3, IL-13, LLT1, AICL, DNAM-1, and NKp80. In some embodiments, the second antigen binding domain is an IL-3 domain. In some embodiments, the IL-3 domain is at least about 85% (eg, at least about 90%, 92%, 95%, 98%, or 99%) sequence identity.

In some embodiments according to any one of the chimeric receptors or multispecific chimeric receptors described above, the NKG2D domain, or the first NKG2D domain and/or the second NKG2D domain, is SEQ ID NO: 7 or 8, or at least about 85% (e.g., at least about 90%, 92%, 95%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 7 or 8; Including variants of it.

In some embodiments according to any one of the chimeric receptors or multispecific chimeric receptors described above, the transmembrane domains include CD8α, CD4, CD28, 4-1BB, CD80, CD86, CD152, and PD1. derived from a molecule selected from the group consisting of. In some embodiments, the transmembrane domain comprises the amino acid sequence of SEQ ID NO: 4 or 45.

In some embodiments according to any one of the chimeric receptors or multispecific chimeric receptors described above, the intracellular signaling domain comprises the primary intracellular signaling domain of the immune effector cell. In some embodiments, the primary intracellular signaling domain is derived from CD3ζ. In some embodiments, the primary intracellular signaling domain comprises the amino acid sequence of SEQ ID NO:6.

In some embodiments according to any one of the chimeric receptors or multispecific chimeric receptors described above, the intracellular signaling domain comprises a costimulatory signaling domain. In some embodiments, the costimulatory signaling domain is of CD27, CD28, 4-1BB, OX40, ICOS, CD30, CD40, CD3, LFA-1, CD2, CD7, LIGHT, NKG2C, B7-H3, CD83. a costimulatory molecule selected from the group consisting of a ligand, and a combination thereof. In some embodiments, the costimulatory signaling domain comprises the cytoplasmic domain of CD28 and/or the cytoplasmic domain of 4-1BB. In some embodiments, the costimulatory signaling domain comprises the amino acid sequence of SEQ ID NO:5.

In some embodiments according to any one of the chimeric receptors or multispecific chimeric receptors described above, the chimeric receptor or multispecific chimeric receptor comprises the C-terminus of the extracellular domain and the transmembrane domain. It further includes a hinge region located between the N-terminus and the N-terminus. In some embodiments, the hinge region is derived from CD8α. In some embodiments, the hinge region comprises the amino acid sequence of SEQ ID NO:3.

In some embodiments, one or more mononucleotides comprising a nucleic acid sequence encoding one or more of the polypeptide chains in any one of the chimeric receptors or multispecific chimeric receptors described above. A separated nucleic acid is provided.

One aspect of the present application provides a dual chimeric receptor system comprising (i) a first chimeric receptor and (ii) a second chimeric receptor, wherein (i) the first chimeric receptor is a first chimeric receptor; one polypeptide chain and a second polypeptide chain, each of the first polypeptide chain and the second polypeptide chain comprising (a) a first extracellular domain comprising an NKG2D domain; (b) a first transmembrane domain; and (c) a first intracellular signaling domain; (ii) the second chimeric receptor comprises: (a) a second extracellular domain comprising a second antigen-binding domain , and (b) a third polypeptide chain comprising a second transmembrane domain. In some embodiments, the second chimeric receptor further comprises a second intracellular signaling domain.

One aspect of the present application provides a dual chimeric receptor system comprising (i) a first chimeric receptor and (ii) a second chimeric receptor, wherein (i) the first chimeric receptor is ( a) a first extracellular domain comprising a first NKG2D domain and a second NKG2D domain; (b) a first transmembrane domain; and (c) a first intracellular signaling domain. (ii) the second chimeric receptor comprises (a) a second extracellular domain comprising a second antigen binding domain; and (b) a second transmembrane domain. It has a second polypeptide chain. In some embodiments, the second chimeric receptor further comprises a second intracellular signaling domain.

In some embodiments according to any one of the dual chimeric receptor systems described above, the second antigen binding domain is an antibody fragment. In some embodiments, the antibody fragment is CD19, CD20, CD22, CD33, CD38, BCMA, CS1, ROR1, GPC3, CD123, CD138, c-Met, EGFR, EGFRvIII, HER2, HER3, GD-2, NY - specifically binds to an antigen selected from the group consisting of ESO-1, MAGE A3, and glycolipid F77. In some embodiments, the second antigen binding domain is a ligand or a ligand binding domain. In some embodiments, the ligand or ligand binding domain is derived from a molecule selected from the group consisting of NKG2A, NKG2C, NKG2F, IL-3, IL-13, LLT1, AICL, DNAM-1, and NKp80. In some embodiments, the second antigen binding domain is an IL-3 domain. In some embodiments, the IL-3 domain is at least about 85% (e.g., at least about 90%, 92%, 95%, 98%) the amino acid sequence of SEQ ID NO: 9, or %, or 99%) sequence identity.

In some embodiments according to any one of the dual chimeric receptor systems described above, the NKG2D domain, or the first NKG2D domain and/or the second NKG2D domain, comprises the amino acid sequence of SEQ ID NO: 7 or 8; or a variant thereof that has at least about 85% (e.g., at least about 90%, 92%, 95%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 7 or 8. .

In some embodiments according to any one of the dual chimeric receptor systems described above, the first and/or second transmembrane domain is CD8α, CD4, CD28, 4-1BB, CD80, CD86, It is derived from a molecule selected from the group consisting of CD152, and PD1. In some embodiments, the first and/or second transmembrane domain comprises the amino acid sequence of SEQ ID NO: 4 or 45.

In some embodiments according to any one of the dual chimeric receptor systems described above, the first and/or second intracellular signaling domain comprises a primary intracellular signaling domain of an immune effector cell. . In some embodiments, the primary intracellular signaling domain is derived from CD3ζ. In some embodiments, the primary intracellular signaling domain comprises the amino acid sequence of SEQ ID NO:6.

In some embodiments according to any one of the dual chimeric receptor systems described above, the first and/or second intracellular signaling domain comprises a costimulatory signaling domain. In some embodiments, the costimulatory signaling domain is of CD27, CD28, 4-1BB, OX40, CD30, ICOS, CD40, CD3, LFA-1, CD2, CD7, LIGHT, NKG2C, B7-H3, CD83. a costimulatory molecule selected from the group consisting of a ligand, and a combination thereof. In some embodiments, the costimulatory signaling domain comprises the cytoplasmic domain of CD28 and/or the cytoplasmic domain of 4-1BB. In some embodiments, the costimulatory signaling domain comprises the amino acid sequence of SEQ ID NO:5.

In some embodiments according to any one of the dual chimeric receptor systems described above, the first and/or second chimeric receptor has a C-terminus of the extracellular domain and an N-terminus of the transmembrane domain. It further includes a hinge region located therebetween. In some embodiments, the hinge region is derived from CD8α. In some embodiments, the hinge region comprises the amino acid sequence of SEQ ID NO:3.

In some embodiments, one or more isolated nucleic acids comprising a nucleic acid sequence encoding one or more polypeptide chains in any one of the dual chimeric receptor systems described above are provided. In some embodiments, an isolated nucleic acid is provided comprising a first nucleic acid sequence encoding a first chimeric receptor and a second nucleic acid sequence encoding a second chimeric receptor, , the first nucleic acid sequence is operably linked to the second nucleic acid sequence via a third nucleic acid sequence encoding a self-cleavable peptide. In some embodiments, the self-cleaving peptide is a T2A, P2A, or F2A peptide.

In some embodiments, for an amino acid sequence selected from the group consisting of SEQ ID NO: 16-20 and SEQ ID NO: 33-35, or an amino acid sequence selected from the group consisting of SEQ ID NO: 16-20 and 33-35. Chimeric receptors are provided that include variants thereof that have at least about 85% (eg, at least about 90%, 92%, 95%, 98%, or 99%) sequence identity. In some embodiments, a dual chimeric receptor system is provided that includes a first chimeric receptor and a second chimeric receptor, wherein the first chimeric receptor has the amino acid sequence of SEQ ID NO: 34, or a variant thereof that has at least about 85% (e.g., at least about 90%, 92%, 95%, 98%, or 99%) sequence identity to the amino acid sequence of number 34; The body has at least about 85% (e.g., at least about 90%, 92%, 95%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 41, or to the amino acid sequence of SEQ ID NO: 41. , including its variants. In some embodiments, a dual chimeric receptor system is provided that includes a first chimeric receptor and a second chimeric receptor, wherein the first chimeric receptor has the amino acid sequence of SEQ ID NO: 35, or a variant thereof that has at least about 85% (e.g., at least about 90%, 92%, 95%, 98%, or 99%) sequence identity to the amino acid sequence of number 35; The body has at least about 85% (e.g., at least about 90%, 92%, 95%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 42, or to the amino acid sequence of SEQ ID NO: 42. , including its variants. In some embodiments, the amino acid sequence of SEQ ID NO: 36 or 37, or at least about 85% (e.g., at least about 90%, 92%, 95%, 98%, or 99%) sequence identity, including variants thereof, are provided. In some embodiments, for a nucleic acid sequence selected from the group consisting of SEQ ID NO: 21-27 and SEQ ID NO: 38-40, or a nucleic acid sequence selected from the group consisting of SEQ ID NO: 21-27 and 38-40. Provided are isolated nucleic acids comprising variants thereof that have at least about 85% (eg, at least about 90%, 92%, 95%, 98%, or 99%) sequence identity.

In some embodiments, one or more vectors are provided encoding any one or more of the isolated nucleic acids described above. In some embodiments, the vector is a lentiviral vector.

Another aspect of the present application is a modified immune effector cell comprising any one of the chimeric receptors, multispecific chimeric receptors, or dual chimeric receptor systems, isolated nucleic acids, or vectors described above. I will provide a. In some embodiments, the immune effector cells are T cells, NK cells, peripheral blood mononuclear cells (PBMCs), hematopoietic stem cells, pluripotent stem cells, or embryonic stem cells. In some embodiments, the immune effector cell is a T cell.

In some embodiments, a pharmaceutical composition is provided comprising any one of the modified immune effector cells described above and a pharmaceutically acceptable carrier.

Another aspect of the present application provides a method of treating cancer in an individual, comprising administering to the individual an effective amount of any one of the pharmaceutical compositions described above. In some embodiments, the cancer is multiple myeloma, acute lymphoblastic leukemia, or chronic lymphocytic leukemia.

Further provided are uses comprising any one of the chimeric receptors, multispecific chimeric receptors, dual chimeric receptor systems, engineered immune effector cells, isolated nucleic acids, or vectors described above. , kits, articles of manufacture.

Figure 2 shows a schematic diagram of an exemplary bispecific chimeric receptor (LIC2001) comprising a single polypeptide chain containing an IL-3 domain and two NKG2D domains. A positively charged residue (R) is engineered at the N-terminus of the first NKG2D domain (e.g., a reverse NKG2D domain), and a negatively charged residue (D) is engineered at the C-terminus of the second NKG2D domain. Engineered at the terminal end (eg, forward NKG2D domain). The engineered R and D residues form salt bridges with each other to promote dimer formation.

Figure 2 shows a schematic diagram of an exemplary bispecific chimeric receptor (LIC2001-1) that includes a single polypeptide chain that includes an IL-3 domain and two NKG2D domains.

Figure 2 shows a schematic diagram of an exemplary bispecific chimeric receptor comprising two polypeptide chains each containing an IL-3 domain, a leucine zipper motif, and an NKG2D domain. A leucine zipper motif in each polypeptide chain promotes dimer formation. Additionally, NKG2D domains can be cross-linked to each other by disulfide bonds. LIC2002 contains an IL-3 domain, a leucine zipper motif, and an inverted NKG2D domain. LIC2002-2 contains an IL-3 domain, a leucine zipper motif, and a forward NKG2D domain.

Figure 2 shows a schematic diagram of an exemplary bispecific chimeric receptor (LIC2002-1) comprising two polypeptide chains each containing an IL-3 domain and an NKG2D domain. NKG2D domains are cross-linked to each other by disulfide bonds.

Figure 2 shows a schematic diagram of an exemplary dual chimeric receptor system (LIC2003) comprising a first chimeric receptor targeting NKG2D ligand and a second chimeric receptor targeting CD123. The first chimeric receptor comprises a polypeptide chain that includes two NKG2D domains, where the two NKG2D domains can be cross-linked to each other by disulfide bonds. The second chimeric receptor contains an IL-3 domain. The second chimeric receptor may or may not contain an intracellular signaling domain.

Figure 2 shows a schematic diagram of an exemplary dual chimeric receptor system (LIC2004) comprising a first chimeric receptor targeting NKG2D ligand and a second chimeric receptor targeting CD123. The first chimeric receptor comprises two polypeptide chains each containing a single NKG2D domain, where the NKG2D domains are cross-linked to each other by disulfide bonds. The second chimeric receptor contains an IL-3 domain. The second chimeric receptor may or may not contain an intracellular signaling domain. This figure shows an exemplary second IL-3 chimeric receptor that does not have an intracellular signaling domain.

Expression of NKG2D×IL-3 chimeric receptor constructs (LIC2004 and LIC2002-2) in engineered T cells as measured by flow cytometry.

Figure 2 shows in vitro cytotoxic activity of engineered T cells expressing various NKG2DxIL-3 chimeric receptor constructs against tumor cells K562-CD123-Luc. Figure 2 shows the in vitro cytotoxic activity of engineered T cells expressing various NKG2DxIL-3 chimeric receptor constructs against tumor cells K562-Luc. Figure 2 shows in vitro cytotoxic activity of engineered T cells expressing various NKG2DxIL-3 chimeric receptor constructs against tumor cells KG1-Luc.

A plot comparing the dose-dependent cytotoxic activity of engineered T cells expressing various chimeric receptor constructs against K562-CD123-Luc is shown.

Figure 2 shows the cytotoxic activity of engineered T cells expressing various constructs against tumor cells K562-CD123-Luc. Figure 2 shows the cytotoxic activity of engineered T cells expressing various constructs against tumor cells K562-Luc. "NKG2D-CD123 binder" refers to engineered T cells expressing the LIC2004 dual chimeric receptor system. "NKG2D" refers to engineered T cells that express only the chimeric receptor containing the NKG2D domain (ie, LIC2004-1) of the LIC2004 dual chimeric receptor system. "CD123 binder" refers to an engineered T cell that expresses only the chimeric receptor containing the IL-3 domain of the LIC2004 dual chimeric receptor system.

Figure 2 shows the inhibitory effect of MICA (cognate ligand of NKG2D) on the killing of K562-CD123-Luc and K562-Luc cells by engineered T cells expressing the NKG2D x IL-3 chimeric receptor construct. We show that BSA has no significant inhibitory effect on the killing of K562-CD123-Luc and K562-Luc cells by engineered T cells expressing the NKG2D×IL-3 chimeric receptor construct.

Figure 3 shows the cytotoxic activity of engineered T cells expressing LIC2004 and LIC2002-2 constructs against K562 and K562-CD123-Luc tumor cells.

Figure 3 shows the secretion levels of IFNγ by co-culture of engineered T cells expressing LIC2002-2, LIC2004, and LIC2004-1 constructs with the K562-CD123-Luc cell line. Figure 3 shows the secretion levels of IFNγ by co-culture of engineered T cells expressing LIC2002-2, LIC2004, and LIC2004-1 constructs with the K562-Luc cell line. Figure 3 shows the secretion levels of IFNγ by co-culture of engineered T cells expressing LIC2002-2, LIC2004, and LIC2004-1 constructs with the KG1-Luc cell line.

The present application provides multispecific (e.g., bispecific) chimeric receptors and dual chimeric receptor systems that target an NKG2D ligand and a second antigen, such as CD123 (e.g., an IL-3 domain). do. In some embodiments, unlike antibody-based CARs, the chimeric receptors and dual chimeric receptor systems described herein provide high affinity and Take advantage of specificity. NKG2D ligands are expressed only on stressed cells, especially tumor cells. Engineered immune cells expressing the NKG2D chimeric receptor and dual chimeric receptor systems described herein have improved antitumor capacity and provide useful therapeutic agents for anticancer therapy. do.

Accordingly, one aspect of the present application provides (a) an extracellular domain comprising an NKG2D domain and a second antigen binding domain (e.g., a CD123-targeting binding domain), (b) a transmembrane domain, and (c) Multispecific chimeric receptors are provided that include intracellular signaling domains.

In some embodiments, (a) an extracellular domain comprising a second antigen binding domain (e.g., an IL-3 domain), a first NKG2D domain, and a second NKG2D domain; (b) a transmembrane domain. and (c) a polypeptide chain comprising an intracellular signaling domain.

In some embodiments, multispecific chimeric receptors are provided that include a first polypeptide chain and a second polypeptide chain, each polypeptide chain comprising (a) an NKG2D domain and a second antigen. (b) a transmembrane domain; and (c) an intracellular signaling domain. In some embodiments, the extracellular domain further comprises a dimerization motif, such as a leucine zipper.

In another aspect, a dual chimeric receptor system is provided comprising (i) a first chimeric receptor and (ii) a second chimeric receptor, wherein (i) the first chimeric receptor comprises (a) a first extracellular domain comprising an NKG2D domain, (b) a first transmembrane domain, and (c) a first intracellular signaling domain; (ii) the second chimeric receptor comprises: (a) a second extracellular domain comprising a second antigen binding domain (e.g., an IL-3 domain); (b) a second transmembrane domain; and optionally (c) a second intracellular signaling domain. . In some embodiments, the first chimeric receptor comprises a single polypeptide chain, wherein the first extracellular domain comprises a first NKG2D domain and a second NKG2D domain. In some embodiments, the first chimeric receptor comprises a first polypeptide chain and a second polypeptide chain, each polypeptide chain comprising: (a) a first cell comprising an NKG2D domain; (b) a first transmembrane domain; and (c) a first intracellular signaling domain.

Engineered immune effector cells (e.g., T cells), pharmaceutical compositions, kits, articles of manufacture, and engineered immune effector cells containing chimeric receptors, multispecific chimeric receptors, or dual chimeric receptor systems are used to treat cancer. Also described herein are methods of treating.
I. definition

As used herein, "chimeric receptor" refers to a genetically engineered receptor that directs one or more specific polypeptide interactions to T cells through antigen-antibody interactions or ligand-receptor binding. can be used to transplant immune effector cells such as Some chimeric receptors are also known as "chimeric antigen receptors," "artificial T cell receptors," "chimeric T cell receptors," or "chimeric immunoreceptors." In some embodiments, the chimeric receptor has an extracellular antigen-binding domain specific for one or more antigens (e.g., a tumor antigen), a transmembrane domain, and an intracellular domain for T cells and/or other receptors. Contains a signaling domain. In some embodiments, the extracellular antigen binding domain comprises at least one domain derived from a ligand or an extracellular domain of a receptor, where the ligand or receptor is a cell surface antigen, such as a tumor antigen. be.

"NKG2D chimeric receptor" refers to a chimeric receptor that has an extracellular domain that includes one or more binding domains (eg, NKG2D domains) specific for NKG2D ligands. "NKG2D x IL-3 chimeric receptor" has an extracellular domain that includes a binding domain specific for NKG2D ligand (e.g., NKG2D domain) and a binding domain specific for CD123 (e.g., IL-3 domain) Refers to chimeric receptors.

As used herein, "NKG2D domain" refers to a functional fragment in the extracellular domain of NKG2D, which functional fragment is specific for one or more NKG2D ligands upon dimerization of the NKG2D domain. can be combined with Exemplary human NKG2D ligands include, but are not limited to, MICA, MICB, and ULBP molecules.

As used herein, "IL-3 domain" refers to a functional fragment of IL-3 (including full-length IL-3), which includes CD123, e.g., the IL-3R complex. and/or may specifically bind to the IL-3RA subunit.

As used herein, the terms "target," "specifically bind," "specifically recognize," or "specific for" refer to a target and an antigen-binding protein (e.g., An interaction that determines the presence of a target in the presence of a heterogeneous population of molecules, including biological molecules do. For example, an antigen-binding protein that specifically binds to a target is an antigen-binding protein that binds to this target with higher affinity, avidity, more easily, and/or for a longer period of time than it binds to other targets. be. In some embodiments, the degree of binding of the antigen binding protein to an unrelated target is less than about 10% of the binding of the antigen binding protein to the target, as measured, for example, by radioimmunoassay (RIA). In some embodiments, the antigen binding protein that specifically binds the target has a dissociation constant (Kd) of ≦1 μM, ≦100 nM, ≦10 nM, ≦1 nM, or ≦0.1 nM. In some embodiments, the antigen binding protein specifically binds an epitope on a protein that is conserved in a protein from a foreign species. In some embodiments, specific binding can include, but does not require, exclusive binding.

The term "specificity" refers to the selective recognition of an antigen binding protein (eg, antigen binding domain, ligand, or chimeric receptor) for a particular epitope of an antigen. As used herein, the term "multispecific" means that an antigen binding protein (e.g., a chimeric receptor) has two or more antigen binding sites, at least two of which bind different antigens. As used herein, the term "bispecific" means that an antigen binding protein (eg, a chimeric receptor) has two different antigen binding specificities.

"Binding affinity" generally refers to the sum of noncovalent interactions between a single binding site of a molecule (e.g., an antigen-binding domain, a ligand, or a chimeric receptor) and its binding partner (e.g., an antigen). Refers to strength. Unless otherwise indicated, "binding affinity," as used herein, refers to an inherent binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antigen-binding domain and an antigen). Refers to affinity. The affinity of molecule X for its partner Y can generally be expressed by the dissociation constant (Kd). Affinity can be measured by common methods known in the art, including those described herein. Antibodies with low affinity generally tend to bind antigen slowly and dissociate easily, whereas antibodies with high affinity generally bind antigen more quickly and tend to remain bound longer. . Various methods of measuring binding affinity are known in the art, any of which can be used for the purposes of this application.

The term "antibody" refers to monoclonal antibodies (including full-length four-chain antibodies or full-length heavy chain-only antibodies with an immunoglobulin Fc region), antibody compositions with polyepitopic specificity, multispecific antibodies (e.g. , bispecific antibodies, bispecific antibodies, single chain molecules), and antibody fragments (eg, Fab, F(ab') ₂ , and Fv). Antibodies contemplated herein include single domain antibodies, such as heavy chain only antibodies.

An "antibody fragment" comprises a portion of an intact antibody, preferably the antigen binding region and/or variable region of an intact antibody. Examples of antibody fragments include Fab, Fab', F(ab') ₂ , and Fv fragments; bispecific antibodies; linear antibodies (U.S. Pat. No. 5,641,870, Example 2; Zapata et al. Al., Protein Eng. 8(10):1057-1062 [1995]); single chain antibody molecules; single domain antibodies (e.g. V _H H), as well as multispecific antibodies formed from antibody fragments. can be mentioned.

A "single chain Fv", also abbreviated as "sFv" or "scFv", is an antibody fragment that comprises V _H and V _L antibody domains connected into a single polypeptide chain. Preferably, the sFv polypeptide further comprises a polypeptide linker between the V _H and V _L domains that enables the sFv to form the desired structure for antigen binding. For a review of sFv, see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds. , Springer-Verlag, New York, pp. 269-315 (1994).

"Percent (%) amino acid sequence identity" and "homology" to a peptide, polypeptide, or antibody sequence refers to the alignment of the sequences and the introduction of gaps, if necessary, to achieve maximum percent sequence identity. It is then defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in the sequence of a particular peptide or polypeptide, without considering any conservative substitutions as part of sequence identity. Alignments for the purpose of determining percent amino acid sequence identity may be performed in a variety of ways within the skill of the art, such as BLAST, BLAST-2, ALIGN, or MEGALIGN™ (DNASTAR Inc.) software. This can be accomplished using available computer software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms necessary to achieve maximum alignment over the full length of the sequences being compared.

An "isolated" nucleic acid molecule encoding a chimeric receptor or dual chimeric receptor system described herein is one that is identified and separated from at least one contaminating nucleic acid molecule with which it is normally associated in the environment in which it was produced. It is a nucleic acid molecule. Preferably, isolated nucleic acids are free of all components associated with the production environment. Isolated nucleic acid molecules encoding polypeptides and antibodies herein are in a form other than that found in nature or configuration. Isolated nucleic acid molecules are thus distinguished from nucleic acids encoding the polypeptides and antibodies herein that naturally occur within a cell.

The term "control sequences" refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism. Control sequences suitable for prokaryotes include, for example, a promoter, optionally an operator sequence, and a ribosome binding site. Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers.

Nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, a presequence or secretion leader DNA is operably linked to the DNA of a polypeptide when expressed as a preprotein involved in secretion of the polypeptide; a promoter or enhancer influences transcription of the sequence. or a ribosome binding site is operably linked to a coding sequence if the ribosome binding site is positioned to facilitate translation. Generally, "operably linked" means that the DNA sequences being joined are contiguous, and in the case of a secretory leader, contiguous and in the process of being decoded. However, enhancers do not have to be in close proximity. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, synthetic oligonucleotide adapters or linkers are used according to conventional practice.

The term "vector" as used herein refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes vectors that are integrated into the genome of the host cell into which they are introduced, as well as vectors as self-replicating nucleic acid structures. Certain vectors are capable of directing the expression of nucleic acids to which they are operably linked. Such vectors are referred to herein as "expression vectors."

As used herein, the term "autologous" is intended to refer to any material derived from the same individual that is later reintroduced to that individual.

"Homologous" refers to grafts that are derived from different individuals of the same species.

The terms "transfected" or "transformed" or "transduced" as used herein refer to the process by which exogenous nucleic acid is transferred or introduced into a host cell. A "transfected" or "transformed" or "transduced" cell is a cell that has been transfected, transformed, or transduced with an exogenous nucleic acid. The cells include the primary subject cell and its progeny.

As used herein, the expressions "cell," "cell line," and "cell culture" are used interchangeably and all such designations include progeny. Thus, the terms "transfectant" and "transfected cell" include the primary subject cell and cultures derived therefrom, regardless of the number of transfers. It is also understood that all progeny may not be exactly identical in DNA content due to deliberate or accidental mutations. Variant progeny that have the same function or biological activity as screened for in the originally transformed cell are included.

As used herein, "treatment" or "treating" is a technique for obtaining beneficial or desired results, including clinical results. For purposes of the present invention, beneficial or desirable clinical outcomes include, but are not limited to, one or more of the following: alleviation of one or more symptoms caused by the disease; reducing the severity, stabilizing the disease (e.g., preventing or slowing the worsening of the disease), preventing or slowing the spread of the disease (e.g., metastasis), preventing or slowing the recurrence of the disease, slowing or slowing the progression of the disease. cure, ameliorate the condition, provide (partial or complete) remission of the disease, reduce the dose of one or more drugs needed to treat the disease, slow disease progression, improve quality of life , and/or prolonging survival. Further encompassed by "treatment" is the reduction of the pathological consequences of cancer. The methods of the present application contemplate any one or more of these aspects of treatment.

As used herein, an "individual" or "subject" includes a human, a bovine, an equid, a feline, a canine, a rodent, or a primate. refers to mammals, including but not limited to. In some embodiments, the individual is a human.

As used herein, the term "effective amount" of an agent, such as a modified immune effector cell or pharmaceutical composition thereof, is used to treat an identified disorder, condition, or disease (e.g., to treat one of its symptoms). or more than one). In the context of cancer, an effective amount is capable of shrinking a tumor and/or decreasing the growth rate of a tumor (e.g., inhibiting tumor growth) or otherwise preventing or slowing undesirable cell proliferation. Contains sufficient amount. In some embodiments, an effective amount is an amount sufficient to retard development. In some embodiments, an effective amount is an amount sufficient to prevent or delay recurrence. An effective amount may be administered in one or more administrations. An effective amount of the drug or composition may (i) reduce the number of cancer cells, (ii) reduce tumor size, or (iii) inhibit or delay to some extent the invasion of cancer cells into peripheral organs. (iv) may inhibit tumor metastasis (i.e. may slow to some extent, preferably may stop); (v) may inhibit tumor growth; (vi) may inhibit tumor development; and/or may prevent or delay recurrence and/or (vii) may alleviate to some extent one or more of the symptoms associated with the cancer.

As used herein, "delaying" cancer development means postponing, preventing, slowing, delaying, stabilizing, and/or prolonging the development of the disease. . This delay can vary in length depending on the history of the disease and/or the individual being treated. As will be appreciated by those skilled in the art, sufficient or significant delay may in fact encompass prevention in that the individual does not develop the disease. A method of "delaying" cancer development reduces the likelihood of disease development in a given time frame and/or reduces the extent of disease in a given time frame compared to not using the method. This is a method to reduce Such comparisons are typically based on clinical studies using statistically significant numbers of individuals. Cancer development can be assessed using standard methods including, but not limited to, computed axial tomography (CAT scan), magnetic resonance imaging (MRI), abdominal ultrasound, coagulation studies, arteriography, or biopsy. may be detectable using Development may also refer to the progression of cancer, which may be undetectable at the beginning of the disease, and includes development, recurrence, and onset.

It is understood that the embodiments of the present application described herein include embodiments "consisting of" and/or "consisting essentially of."

References herein to "about" a value or parameter include (and describe) variations directed at that value or parameter itself. For example, descriptions that refer to "about X" include descriptions of "X."

As used herein, reference to a value or parameter with "not" generally means and describes "other than" the value or parameter. For example, the method is not used to treat type X cancer means that the method is used to treat cancer types other than X.

As used herein, the term "about X to Y" has the same meaning as "about X to about Y."

As used in this specification and the appended claims, the singular forms "a,""an," and "the" include plural referents unless the context clearly dictates otherwise.
II. Multispecific chimeric receptors and dual chimeric receptor systems

This application provides chimeric receptors and chimeric receptor systems that target NKG2D ligands. One aspect of the present application provides a multispecific chimeric receptor that includes an extracellular domain that includes an NKG2D domain and a second antigen binding domain, such as a CD123 binding domain, eg, an IL-3 domain.

In some embodiments, chimeric receptors are provided that include (a) an extracellular domain that includes an NKG2D domain, (b) a transmembrane domain, and (c) an intracellular signaling domain. In some embodiments, the transmembrane domain is derived from a molecule selected from the group consisting of CD8α, CD4, CD28, CD137, CD80, CD86, CD152, and PD1. In some embodiments, the intracellular signaling domain comprises the primary intracellular signaling domain of an immune effector cell (eg, a T cell). In some embodiments, the primary intracellular signaling domain is derived from CD3ζ. In some embodiments, the intracellular signaling domain comprises a costimulatory signaling domain. In some embodiments, the costimulatory signaling domain is of CD27, CD28, 4-1BB, OX40, CD30, ICOS, CD40, CD3, LFA-1, CD2, CD7, LIGHT, NKG2C, B7-H3, CD83. a costimulatory molecule selected from the group consisting of a ligand, and a combination thereof. In some embodiments, the chimeric receptor further comprises a hinge region (eg, a CD8α hinge region) located between the C-terminus of the extracellular domain and the N-terminus of the transmembrane domain. In some embodiments, the chimeric receptor comprises, from N-terminus to C-terminus, a first NKG2D domain, a peptide linker, a second NKG2D domain, a transmembrane domain (CD8α), a costimulatory domain derived from 4-1BB. , and the primary signaling domain derived from CD3ζ. In some embodiments, the NKG2D domain comprises the amino acid sequence of SEQ ID NO:8.

In some embodiments, chimeric receptors are provided that include a first polypeptide chain and a second polypeptide chain, each polypeptide chain having (a) an NKG2D domain and a dimerization motif (e.g. (b) a transmembrane domain; and (c) an intracellular signaling domain. In some embodiments, the NKG2D domain of the first polypeptide chain is cross-linked to the NKG2D domain of the second polypeptide chain by one or more disulfide bonds. In some embodiments, the transmembrane domain is derived from a molecule selected from the group consisting of CD8α, CD4, CD28, CD137, CD80, CD86, CD152, and PD1. In some embodiments, the intracellular signaling domain comprises the primary intracellular signaling domain of an immune effector cell (eg, a T cell). In some embodiments, the primary intracellular signaling domain is derived from CD3ζ. In some embodiments, the intracellular signaling domain comprises a costimulatory signaling domain. In some embodiments, the costimulatory signaling domain is of CD27, CD28, 4-1BB, OX40, CD30, ICOS, CD40, CD3, LFA-1, CD2, CD7, LIGHT, NKG2C, B7-H3, CD83. A costimulatory molecule selected from the group consisting of a ligand, and a combination thereof. In some embodiments, each polypeptide chain further comprises a hinge region (e.g., a CD8α hinge region) located between the C-terminus of the first extracellular domain and the N-terminus of the first transmembrane domain. . In some embodiments, each polypeptide chain further comprises a signal peptide (eg, CD8α signal peptide) located at the N-terminus of each polypeptide chain.

In some embodiments, the chimeric receptor comprises (a) an extracellular domain comprising a first NKG2D domain and a second NKG2D domain, (b) a transmembrane domain, and (c) an intracellular signaling domain. provided. In some embodiments, the transmembrane domain is derived from a molecule selected from the group consisting of CD8α, CD4, CD28, CD137, CD80, CD86, CD152, and PD1. In some embodiments, the intracellular signaling domain comprises the primary intracellular signaling domain of an immune effector cell (eg, a T cell). In some embodiments, the primary intracellular signaling domain is derived from CD3ζ. In some embodiments, the intracellular signaling domain comprises a costimulatory signaling domain. In some embodiments, the costimulatory signaling domain is of CD27, CD28, 4-1BB, OX40, CD30, ICOS, CD40, CD3, LFA-1, CD2, CD7, LIGHT, NKG2C, B7-H3, CD83. A costimulatory molecule selected from the group consisting of a ligand, and a combination thereof. In some embodiments, the chimeric receptor further comprises a hinge region (eg, a CD8α hinge region) located between the C-terminus of the extracellular domain and the N-terminus of the transmembrane domain. In some embodiments, the chimeric receptor comprises, from N-terminus to C-terminus, a first NKG2D domain, a peptide linker, a second NKG2D domain, a transmembrane domain (CD8α), a costimulatory domain derived from 4-1BB. , and the primary signaling domain derived from CD3ζ. In some embodiments, the NKG2D domain comprises the amino acid sequence of SEQ ID NO:8. In some embodiments, about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, relative to the amino acid sequence of SEQ ID NO: 33; Chimeric receptors are provided that include amino acid sequences having at least one of 96%, 97%, 98%, 99%, or 100% sequence identity. In some embodiments, about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, Isolated nucleic acid sequences are provided that include nucleic acid sequences having at least one of 96%, 97%, 98%, 99%, or 100% sequence identity.

In some embodiments, (a) an extracellular domain comprising an NKG2D domain and a second antigen binding domain (e.g., an IL-3 domain), (b) a transmembrane domain, and (c) an intracellular signaling domain. Multispecific (e.g., bispecific) chimeric receptors are provided that include. In some embodiments, the second antigen binding domain is CD19, CD20, CD22, CD33, CD38, BCMA, CS1, ROR1, GPC3, CD123, IL-13R, CD138, c-Met, EGFR, EGFRvIII, HER2 , HER3, GD-2, NY-ESO-1, MAGE A3, and glycolipid F77. In some embodiments, the second antigen binding domain is derived from a molecule selected from the group consisting of NKG2A, NKG2C, NKG2F, IL-3, IL-13, LLT1, AICL, DNAM-1, and NKp80. A ligand or a ligand binding domain. In some embodiments, the transmembrane domain is derived from a molecule selected from the group consisting of CD8α, CD4, CD28, CD137, CD80, CD86, CD152, and PD1. In some embodiments, the intracellular signaling domain comprises the primary intracellular signaling domain of an immune effector cell (eg, a T cell). In some embodiments, the primary intracellular signaling domain is derived from CD3ζ. In some embodiments, the intracellular signaling domain comprises a costimulatory signaling domain. In some embodiments, the costimulatory signaling domain is of CD27, CD28, 4-1BB, OX40, CD30, ICOS, CD40, CD3, LFA-1, CD2, CD7, LIGHT, NKG2C, B7-H3, CD83. A costimulatory molecule selected from the group consisting of a ligand, and a combination thereof. In some embodiments, the multispecific chimeric receptor further comprises a hinge region (eg, a CD8α hinge region) located between the C-terminus of the extracellular domain and the N-terminus of the transmembrane domain.

A chimeric receptor (eg, a multispecific chimeric receptor) may include one or more polypeptide chains. In some embodiments, chimeric receptors are monomeric. In some embodiments, the monomeric chimeric receptor includes an extracellular domain that includes a first NKG2D domain and a second NKG2D domain. In some embodiments, the extracellular domain includes, from N-terminus to C-terminus, a second antigen binding domain (eg, an IL-3 domain), a first NKG2D domain, and a second NKG2D domain. In some embodiments, the extracellular domain comprises, from N-terminus to C-terminus, a first NKG2D domain, a second NKG2D domain, and a second antigen binding domain (eg, an IL-3 domain). In some embodiments, a first NKG2D domain is cross-linked to a second NKG2D domain. In some embodiments, the first NKG2D domain includes a first engineered residue at the N-terminus and the second NKG2D domain includes a second engineered residue at the C-terminus. , where the first engineered residue is associated to the second engineered residue, eg, by a disulfide bond or a salt bridge. In some embodiments, the second antigen-binding domain is linked via a peptide linker, such as a peptide linker of about 50 amino acids or less in length, e.g., a peptide linker comprising an amino acid sequence selected from SEQ ID NO: 12-15. fused to the first NKG2D domain.

In some embodiments, a chimeric receptor (eg, a multispecific chimeric receptor) is a dimer, such as a homodimer or a heterodimer. In some embodiments, the dimeric chimeric receptor comprises two polypeptide chains each containing a single NKG2D domain. In some embodiments, a dimeric chimeric receptor comprises two identical polypeptide chains. In some embodiments, a dimeric chimeric receptor comprises two different polypeptide chains. In some embodiments, each polypeptide chain, from N-terminus to C-terminus, includes a second antigen binding domain, an NKG2D domain, a transmembrane domain, and an intracellular signaling domain. In some embodiments, each polypeptide chain, from N-terminus to C-terminus, includes an NKG2D domain, a second antigen binding domain, a transmembrane domain, and an intracellular signaling domain. In some embodiments, the NKG2D domains of each polypeptide chain of a dimeric chimeric receptor are non-covalently associated with each other to form a dimer. In some embodiments, the NKG2D domain of each polypeptide chain of the dimeric chimeric receptor is linked in the extracellular domain, e.g., by a disulfide bond, and/or with a dimerization motif (e.g., a leucine zipper or a cysteine zipper). ), they covalently bind to each other to form dimers. In some embodiments, the second antigen binding domain binds NKG2D via a peptide linker, such as a peptide linker of about 50 amino acids or less in length, e.g., a peptide linker comprising an amino acid sequence selected from SEQ ID NOs: 12-15. merged into the domain. In some embodiments, the second antigen binding domain is fused to the NKG2D domain via a dimerization motif, such as a leucine zipper or a cysteine zipper.

Thus, in some embodiments, (a) an extracellular domain comprising a first NKG2D domain, a second NKG2D domain, and a second antigen binding domain, (b) a transmembrane domain, and (c) Multispecific (eg, bispecific) chimeric receptors are provided that include polypeptide chains that include intracellular signaling domains. In some embodiments, the second antigen binding domain is CD19, CD20, CD22, CD33, CD38, BCMA, CS1, ROR1, GPC3, CD123, IL-13R, CD138, c-Met, EGFR, EGFRvIII, HER2 , HER3, GD-2, NY-ESO-1, MAGE A3, and glycolipid F77. In some embodiments, the second antigen binding domain is derived from a molecule selected from the group consisting of NKG2A, NKG2C, NKG2F, IL-3, IL-13, LLT1, AICL, DNAM-1, and NKp80. A ligand or a ligand binding domain. In some embodiments, the second antigen-binding domain is linked via a peptide linker, such as a peptide linker of about 50 amino acids or less in length, e.g., a peptide linker comprising an amino acid sequence selected from SEQ ID NO: 12-15. fused to a first NKG2D domain or a second NKG2D domain. In some embodiments, the transmembrane domain is derived from a molecule selected from the group consisting of CD8α, CD4, CD28, CD137, CD80, CD86, CD152, and PD1. In some embodiments, the intracellular signaling domain comprises the primary intracellular signaling domain of an immune effector cell (eg, a T cell). In some embodiments, the primary intracellular signaling domain is derived from CD3ζ. In some embodiments, the intracellular signaling domain comprises a costimulatory signaling domain. In some embodiments, the costimulatory signaling domain is of CD27, CD28, 4-1BB, OX40, CD30, ICOS, CD40, CD3, LFA-1, CD2, CD7, LIGHT, NKG2C, B7-H3, CD83. A costimulatory molecule selected from the group consisting of a ligand, and a combination thereof. In some embodiments, the multispecific chimeric receptor further comprises a hinge region (eg, a CD8α hinge region) located between the C-terminus of the extracellular domain and the N-terminus of the transmembrane domain. In some embodiments, the multispecific chimeric receptor further comprises a signal peptide (eg, CD8α signal peptide) located at the N-terminus of the polypeptide chain. In some embodiments, the polypeptide chain, from N-terminus to C-terminus, includes a second antigen binding domain (e.g., an IL-3 domain), a first peptide linker, a first NKG2D domain, a second peptide It contains a linker, a second NKG2D domain, a CD8α hinge region, a CD8α transmembrane domain, a costimulatory signaling domain derived from 4-1BB, and a primary intracellular signaling domain derived from CD3ζ.

In some embodiments, multispecific (e.g., bispecific) chimeric receptors are provided that include a first polypeptide chain and a second polypeptide chain, each polypeptide chain comprising (a) an extracellular domain comprising an NKG2D domain and a second antigen binding domain; (b) a transmembrane domain; and (c) an intracellular signaling domain. In some embodiments, the NKG2D domain of the first polypeptide chain is cross-linked to the NKG2D domain of the second polypeptide chain by one or more disulfide bonds. In some embodiments, the second antigen binding domain is CD19, CD20, CD22, CD33, CD38, BCMA, CS1, ROR1, GPC3, CD123, IL-13R, CD138, c-Met, EGFR, EGFRvIII, HER2 , HER3, GD-2, NY-ESO-1, MAGE A3, and glycolipid F77. In some embodiments, the second antigen binding domain is derived from a molecule selected from the group consisting of NKG2A, NKG2C, NKG2F, IL-3, IL-13, LLT1, AICL, DNAM-1, and NKp80. A ligand or a ligand binding domain. In some embodiments, the second antigen binding domain binds NKG2D via a peptide linker, such as a peptide linker of about 50 amino acids or less in length, e.g., a peptide linker comprising an amino acid sequence selected from SEQ ID NOs: 12-15. merged into the domain. In some embodiments, the transmembrane domain is derived from a molecule selected from the group consisting of CD8α, CD4, CD28, CD137, CD80, CD86, CD152, and PD1. In some embodiments, the intracellular signaling domain comprises the primary intracellular signaling domain of an immune effector cell (eg, a T cell). In some embodiments, the primary intracellular signaling domain is derived from CD3ζ. In some embodiments, the intracellular signaling domain comprises a costimulatory signaling domain. In some embodiments, the costimulatory signaling domain is of CD27, CD28, 4-1BB, OX40, CD30, ICOS, CD40, CD3, LFA-1, CD2, CD7, LIGHT, NKG2C, B7-H3, CD83. A costimulatory molecule selected from the group consisting of a ligand, and a combination thereof. In some embodiments, each polypeptide chain further comprises a hinge region (e.g., a CD8α hinge region) located between the C-terminus of the first extracellular domain and the N-terminus of the first transmembrane domain. . In some embodiments, each polypeptide chain further comprises a signal peptide (eg, CD8α signal peptide) located at the N-terminus of each polypeptide chain. In some embodiments, each of the first polypeptide chain and the second polypeptide chain comprises, from the N-terminus to the C-terminus, a second antigen binding domain (e.g., an IL-3 domain), a peptide linker, a NKG2D domain, the CD8α hinge region, the CD8α transmembrane domain, the costimulatory signaling domain derived from 4-1BB, and the primary intracellular signaling domain derived from CD3ζ.

In some embodiments, multispecific (e.g., bispecific) chimeric receptors are provided that include a first polypeptide chain and a second polypeptide chain, each polypeptide chain comprising (a) an extracellular domain comprising an NKG2D domain, a dimerization motif (e.g., a leucine zipper or a cysteine zipper), and a second antigen-binding domain; (b) a transmembrane domain; and (c) an intracellular signaling domain. include. In some embodiments, the NKG2D domain of the first polypeptide chain is cross-linked to the NKG2D domain of the second polypeptide chain by one or more disulfide bonds. In some embodiments, the second antigen binding domain is CD19, CD20, CD22, CD33, CD38, BCMA, CS1, ROR1, GPC3, CD123, IL-13R, CD138, c-Met, EGFR, EGFRvIII, HER2 , HER3, GD-2, NY-ESO-1, MAGE A3, and glycolipid F77. In some embodiments, the second antigen binding domain is derived from a molecule selected from the group consisting of NKG2A, NKG2C, NKG2F, IL-3, IL-13, LLT1, AICL, DNAM-1, and NKp80. A ligand or a ligand binding domain. In some embodiments, the transmembrane domain is derived from a molecule selected from the group consisting of CD8α, CD4, CD28, CD137, CD80, CD86, CD152, and PD1. In some embodiments, the intracellular signaling domain comprises the primary intracellular signaling domain of an immune effector cell (eg, a T cell). In some embodiments, the primary intracellular signaling domain is derived from CD3ζ. In some embodiments, the intracellular signaling domain comprises a costimulatory signaling domain. In some embodiments, the costimulatory signaling domain is of CD27, CD28, 4-1BB, OX40, CD30, ICOS, CD40, CD3, LFA-1, CD2, CD7, LIGHT, NKG2C, B7-H3, CD83. A costimulatory molecule selected from the group consisting of a ligand, and a combination thereof. In some embodiments, each polypeptide chain further comprises a hinge region (e.g., a CD8α hinge region) located between the C-terminus of the first extracellular domain and the N-terminus of the first transmembrane domain. . In some embodiments, each polypeptide chain further comprises a signal peptide (eg, CD8α signal peptide) located at the N-terminus of each polypeptide chain. In some embodiments, each of the first polypeptide chain and the second polypeptide chain comprises, from the N-terminus to the C-terminus, a second antigen binding domain (e.g., an IL-3 domain), a leucine zipper, a NKG2D domain, the CD8α hinge region, the CD8α transmembrane domain, the costimulatory signaling domain derived from 4-1BB, and the primary intracellular signaling domain derived from CD3ζ.

In some embodiments, the second antigen binding domain specifically binds a cell surface antigen, such as a tumor antigen. Exemplary tumor antigens include CD19, CD20, CD22, CD33, CD38, BCMA, CS1, ROR1, GPC3, CD123, CD138, c-Met, EGFR, EGFRvIII, HER2, HER3, GD-2, NY-ESO- 1, MAGE A3, and Glycolipid F77. In some embodiments, the second antigen binding domain is an antibody fragment, such as a single chain antibody (eg, scFv) or a single domain antibody (eg, VHH). In some embodiments, the second antigen binding domain is an antibody fragment (eg, scFv or VHH) that specifically binds CD123 (eg, an IL-3R or IL-3RA subunit).

In some embodiments, the second antigen binding domain is a ligand. In some embodiments, the second antigen binding domain is a ligand binding domain, such as the extracellular domain of a receptor. Exemplary ligands and receptors include, but are not limited to, NKG2A, NKG2C, NKG2F, IL-3, IL-13, LLT1, AICL, DNAM-1, and NKp80. In some embodiments, the second antigen binding domain is an IL-3 domain.

Thus, in some embodiments, (a) an extracellular domain comprising a first NKG2D domain, a second NKG2D domain, and a CD123 binding domain (e.g., an IL-3 domain); (b) a transmembrane domain; and (c) multispecific (eg, bispecific) chimeric receptors are provided that include a polypeptide chain that includes an intracellular signaling domain. In some embodiments, the CD123 binding domain is linked to the first via a peptide linker, such as a peptide linker of about 50 amino acids or less in length, e.g., a peptide linker comprising an amino acid sequence selected from SEQ ID NOs: 12-15. NKG2D domain or fused to a second NKG2D domain. In some embodiments, the CD123 binding domain is an anti-CD123 antibody fragment (eg, scFv or VHH). In some embodiments, the CD123 binding domain is an IL-3 domain. In some embodiments, the transmembrane domain is derived from a molecule selected from the group consisting of CD8α, CD4, CD28, CD137, CD80, CD86, CD152, and PD1. In some embodiments, the intracellular signaling domain comprises the primary intracellular signaling domain of an immune effector cell (eg, a T cell). In some embodiments, the primary intracellular signaling domain is derived from CD3ζ. In some embodiments, the intracellular signaling domain comprises a costimulatory signaling domain. In some embodiments, the costimulatory signaling domain is of CD27, CD28, 4-1BB, OX40, CD30, ICOS, CD40, CD3, LFA-1, CD2, CD7, LIGHT, NKG2C, B7-H3, CD83. A costimulatory molecule selected from the group consisting of a ligand, and a combination thereof. In some embodiments, the multispecific chimeric receptor further comprises a hinge region (eg, a CD8α hinge region) located between the C-terminus of the extracellular domain and the N-terminus of the transmembrane domain. In some embodiments, the multispecific chimeric receptor further comprises a signal peptide (eg, CD8α signal peptide) located at the N-terminus of the polypeptide chain. In some embodiments, the polypeptide chain, from N-terminus to C-terminus, includes an IL-3 domain, a first peptide linker, a first NKG2D domain, a second peptide linker, a second NKG2D domain, a CD8α hinge. region, the CD8α transmembrane domain, the costimulatory signaling domain derived from 4-1BB, and the primary intracellular signaling domain derived from CD3ζ. Exemplary multispecific chimeric receptors are shown in FIGS. 1A-1B.

In some embodiments, multispecific (e.g., bispecific) chimeric receptors are provided that include a first polypeptide chain and a second polypeptide chain, each polypeptide chain comprising (a) (b) a transmembrane domain; and (c) an intracellular signaling domain. In some embodiments, the NKG2D domain of the first polypeptide chain is cross-linked to the NKG2D domain of the second polypeptide chain by one or more disulfide bonds. In some embodiments, the CD123 binding domain is fused to the NKG2D domain via a peptide linker, such as a peptide linker of about 50 amino acids or less in length, e.g., a peptide linker comprising an amino acid sequence selected from SEQ ID NOs: 12-15. be done. In some embodiments, the CD123 binding domain is an anti-CD123 antibody fragment (eg, scFv or VHH). In some embodiments, the CD123 binding domain is an IL-3 domain. In some embodiments, the transmembrane domain is derived from a molecule selected from the group consisting of CD8α, CD4, CD28, CD137, CD80, CD86, CD152, and PD1. In some embodiments, the intracellular signaling domain comprises the primary intracellular signaling domain of an immune effector cell (eg, a T cell). In some embodiments, the primary intracellular signaling domain is derived from CD3ζ. In some embodiments, the intracellular signaling domain comprises a costimulatory signaling domain. In some embodiments, the costimulatory signaling domain is of CD27, CD28, 4-1BB, OX40, CD30, ICOS, CD40, CD3, LFA-1, CD2, CD7, LIGHT, NKG2C, B7-H3, CD83. A costimulatory molecule selected from the group consisting of a ligand, and a combination thereof. In some embodiments, each polypeptide chain further comprises a hinge region (e.g., a CD8α hinge region) located between the C-terminus of the first extracellular domain and the N-terminus of the first transmembrane domain. . In some embodiments, each polypeptide chain further comprises a signal peptide (eg, CD8α signal peptide) located at the N-terminus of each polypeptide chain. In some embodiments, each of the first polypeptide chain and the second polypeptide chain, from N-terminus to C-terminus, includes an IL-3 domain, a peptide linker, an NKG2D domain, a CD8α hinge region, a CD8α transmembrane domain. , a costimulatory signaling domain derived from 4-1BB, and a primary intracellular signaling domain derived from CD3ζ. An exemplary multispecific chimeric receptor is shown in Figure ID.

In some embodiments, multispecific (e.g., bispecific) chimeric receptors are provided that include a first polypeptide chain and a second polypeptide chain, each polypeptide chain comprising (a) an extracellular domain comprising an NKG2D domain, a dimerization motif (e.g., a leucine zipper or a cysteine zipper), and a CD123 binding domain (e.g., an IL-3 domain); (b) a transmembrane domain; and (c) an intracellular domain. Contains a signaling domain. In some embodiments, the NKG2D domain of the first polypeptide chain is cross-linked to the NKG2D domain of the second polypeptide chain by one or more disulfide bonds. In some embodiments, the CD123 binding domain is an anti-CD123 antibody fragment (eg, scFv or VHH). In some embodiments, the CD123 binding domain is an IL-3 domain. In some embodiments, the transmembrane domain is derived from a molecule selected from the group consisting of CD8α, CD4, CD28, CD137, CD80, CD86, CD152, and PD1. In some embodiments, the intracellular signaling domain comprises the primary intracellular signaling domain of an immune effector cell (eg, a T cell). In some embodiments, the primary intracellular signaling domain is derived from CD3ζ. In some embodiments, the intracellular signaling domain comprises a costimulatory signaling domain. In some embodiments, the costimulatory signaling domain is of CD27, CD28, 4-1BB, OX40, CD30, ICOS, CD40, CD3, LFA-1, CD2, CD7, LIGHT, NKG2C, B7-H3, CD83. A costimulatory molecule selected from the group consisting of a ligand, and a combination thereof. In some embodiments, each polypeptide chain further comprises a hinge region (e.g., a CD8α hinge region) located between the C-terminus of the first extracellular domain and the N-terminus of the first transmembrane domain. . In some embodiments, each polypeptide chain further comprises a signal peptide (eg, CD8α signal peptide) located at the N-terminus of each polypeptide chain. In some embodiments, each of the first polypeptide chain and the second polypeptide chain, from N-terminus to C-terminus, includes an IL-3 domain, a leucine zipper, an NKG2D domain, a CD8α hinge region, a CD8α transmembrane domain. , a costimulatory signaling domain derived from 4-1BB, and a primary intracellular signaling domain derived from CD3ζ. An exemplary multispecific chimeric receptor is shown in Figure 1C.

Exemplary NKG2DxIL-3 chimeric receptors and their sequences are shown in Table 1. For dimeric chimeric receptors with two identical polypeptide chains, the amino acid sequences of the monomeric subunits are shown. In some embodiments, about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, NKG2D x IL-3 chimeric receptor comprising a polypeptide having at least one of 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity is provided. In some embodiments, an NKG2DxIL-3 chimeric receptor is provided that includes an amino acid sequence selected from the group consisting of SEQ ID NOs: 16-20. In some embodiments, about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95% with respect to the amino acid sequence of SEQ ID NO: 33. , 96%, 97%, 98%, 99%, or 100%. In some embodiments, an NKG2D chimeric receptor is provided that includes the amino acid sequence of SEQ ID NO: 33. Also provided is a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 16-20 and 33.

In some embodiments, one or more isolated nucleic acids encoding any of the chimeric receptors or multispecific chimeric receptors provided herein are provided. In some embodiments, where the chimeric receptor is a dimeric chimeric receptor with two identical polypeptide chains, the monomeric subunits of the chimeric receptor encode a single copy of the polypeptide chain. Isolated nucleic acids are provided. In some embodiments, about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92% for a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 21-25 and 38. , 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity is provided. In some embodiments, the isolated nucleic acid is DNA. In some embodiments, the isolated nucleic acid is RNA (eg, mRNA). In some embodiments, one or more vectors are provided that include any one of the chimeric receptors or multispecific chimeric receptor-encoding nucleic acids described above. In some embodiments, the vector is an expression vector. In some embodiments, the vector is a viral vector, such as a lentiviral vector. In some embodiments, the vector is a non-viral vector.
Table 1 Exemplary NKG2DxIL-3 chimeric receptors.
Dual chimeric receptor system

One aspect of the present application provides a dual chimeric receptor system comprising (i) a first chimeric receptor and (ii) a second chimeric receptor, wherein (i) the first chimeric receptor is ( a) a first extracellular domain comprising an NKG2D domain, (b) a first transmembrane domain, and (c) a first intracellular signaling domain; (ii) a second chimeric receptor comprising: a) a second extracellular domain comprising a second antigen binding domain, (b) a second transmembrane domain, and optionally (c) a second intracellular signaling domain. The first chimeric receptor specifically binds the NKG2D ligand and the second chimeric receptor specifically binds a second antigen, such as a tumor antigen, eg, CD123.

Each of the first chimeric receptor and the second chimeric receptor may include one or more polypeptide chains. A dual chimeric receptor system may include any combination of a first chimeric receptor and a second chimeric receptor described herein.

In some embodiments, the first chimeric receptor has (a) a first extracellular domain comprising a first NKG2D domain and a second NKG2D domain, (b) a first transmembrane domain, and (c) comprises a single polypeptide chain that includes a first intracellular signaling domain. In some embodiments, a first NKG2D domain is cross-linked to a second NKG2D domain. In some embodiments, the first NKG2D domain includes a first engineered residue at the N-terminus and the second NKG2D domain includes a second engineered residue at the C-terminus. , where the first engineered residue is associated to the second engineered residue, eg, by a disulfide bond or a salt bridge. In some embodiments, the first transmembrane domain is derived from a molecule selected from the group consisting of CD8α, CD4, CD28, CD137, CD80, CD86, CD152, and PD1. In some embodiments, the first intracellular signaling domain comprises the primary intracellular signaling domain of an immune effector cell (eg, a T cell). In some embodiments, the primary intracellular signaling domain is derived from CD3ζ. In some embodiments, the first intracellular signaling domain comprises a costimulatory signaling domain. In some embodiments, the costimulatory signaling domain is of CD27, CD28, 4-1BB, OX40, CD30, ICOS, CD40, CD3, LFA-1, CD2, CD7, LIGHT, NKG2C, B7-H3, CD83. a costimulatory molecule selected from the group consisting of a ligand, and a combination thereof. In some embodiments, the first chimeric receptor comprises a hinge region (e.g., a CD8α hinge region) located between the C-terminus of the first extracellular domain and the N-terminus of the first transmembrane domain. Including further. In some embodiments, the first chimeric receptor further comprises a signal peptide (eg, a CD8α signal peptide) located at the N-terminus of the polypeptide chain. In some embodiments, the first chimeric receptor comprises, from N-terminus to C-terminus, the first NKG2D domain, the peptide linker, the second NKG2D domain, the CD8α hinge region, the CD8α transmembrane domain, 4-1BB. and a primary intracellular signaling domain derived from CD3ζ. An exemplary first chimeric receptor is shown in FIG. 1E.

In some embodiments, the first chimeric receptor comprises a first polypeptide chain and a second polypeptide chain, each polypeptide chain comprising: (a) a first cell comprising an NKG2D domain; (b) a first transmembrane domain; and (c) a first intracellular signaling domain. In some embodiments, the NKG2D domain of the first polypeptide chain is cross-linked to the NKG2D domain of the second polypeptide chain by one or more disulfide bonds. In some embodiments, the first transmembrane domain is derived from a molecule selected from the group consisting of CD8α, CD4, CD28, CD137, CD80, CD86, CD152, and PD1. In some embodiments, the first intracellular signaling domain comprises the primary intracellular signaling domain of an immune effector cell (eg, a T cell). In some embodiments, the primary intracellular signaling domain is derived from CD3ζ. In some embodiments, the first intracellular signaling domain comprises a costimulatory signaling domain. In some embodiments, the costimulatory signaling domain is of CD27, CD28, 4-1BB, OX40, CD30, ICOS, CD40, CD3, LFA-1, CD2, CD7, LIGHT, NKG2C, B7-H3, CD83. A costimulatory molecule selected from the group consisting of a ligand, and a combination thereof. In some embodiments, each polypeptide chain further comprises a hinge region (e.g., a CD8α hinge region) located between the C-terminus of the first extracellular domain and the N-terminus of the first transmembrane domain. . In some embodiments, each polypeptide chain further comprises a signal peptide (eg, CD8α signal peptide) located at the N-terminus of each polypeptide chain. In some embodiments, each of the first polypeptide chain and the second polypeptide chain comprises, from N-terminus to C-terminus, a co-nucleotide derived from the NKG2D domain, the CD8α hinge region, the CD8α transmembrane domain, 4-1BB. It contains a stimulatory signaling domain, and a primary intracellular signaling domain derived from CD3ζ. An exemplary first chimeric receptor is shown in FIG. 1F.

In some embodiments, the first chimeric receptor comprises a first polypeptide chain and a second polypeptide chain, each polypeptide chain having (a) an NKG2D domain and a dimerization domain. (b) a first transmembrane domain; and (c) a first intracellular signaling domain. In some embodiments, the dimerization domain is located at the N-terminus of the NKG2D domain. In some embodiments, the dimerization domain is located at the C-terminus of the NKG2D domain. In some embodiments, the NKG2D domain of the first polypeptide chain is further cross-linked to the NKG2D domain of the second polypeptide chain by one or more disulfide bonds. In some embodiments, the first transmembrane domain is derived from a molecule selected from the group consisting of CD8α, CD4, CD28, CD137, CD80, CD86, CD152, and PD1. In some embodiments, the first intracellular signaling domain comprises the primary intracellular signaling domain of an immune effector cell (eg, a T cell). In some embodiments, the primary intracellular signaling domain is derived from CD3ζ. In some embodiments, the first intracellular signaling domain comprises a costimulatory signaling domain. In some embodiments, the costimulatory signaling domain is of CD27, CD28, 4-1BB, OX40, CD30, ICOS, CD40, CD3, LFA-1, CD2, CD7, LIGHT, NKG2C, B7-H3, CD83. A costimulatory molecule selected from the group consisting of a ligand, and a combination thereof. In some embodiments, each polypeptide chain further comprises a hinge region (e.g., a CD8α hinge region) located between the C-terminus of the first extracellular domain and the N-terminus of the first transmembrane domain. . In some embodiments, each polypeptide chain further comprises a signal peptide (eg, CD8α signal peptide) located at the N-terminus of each polypeptide chain. In some embodiments, each of the first polypeptide chain and the second polypeptide chain, from N-terminus to C-terminus, includes a leucine zipper, an NKG2D domain, a CD8α hinge region, a CD8α transmembrane domain, a 4-1BB a costimulatory signaling domain derived from CD3ζ, and a primary intracellular signaling domain derived from CD3ζ.

In some embodiments, the second chimeric receptor comprises (a) a second extracellular domain comprising a second antigen binding domain, and (b) a polypeptide chain comprising a second transmembrane domain. . In some embodiments, the second chimeric receptor does not include an intracellular signaling domain. In some embodiments, the second extracellular domain further comprises an NKG2D domain, e.g., the second extracellular domain comprises, from the N-terminus to the C-terminus, an NKG2D domain and a second antigen-binding domain. or comprises a second antigen-binding domain and an NKG2D domain. In some embodiments, the second antigen binding domain is CD19, CD20, CD22, CD33, CD38, BCMA, CS1, ROR1, GPC3, CD123, IL-13R, CD138, c-Met, EGFR, EGFRvIII, HER2 , HER3, GD-2, NY-ESO-1, MAGE A3, and glycolipid F77. In some embodiments, the second antigen binding domain is derived from a molecule selected from the group consisting of NKG2A, NKG2C, NKG2F, IL-3, IL-13, LLT1, AICL, DNAM-1, and NKp80. A ligand or a ligand binding domain. In some embodiments, the second antigen binding domain is a CD123 binding domain, such as an IL-3 domain. In some embodiments, the second transmembrane domain is derived from a molecule selected from the group consisting of CD8α, CD4, CD28, CD137, CD80, CD86, CD152, and PD1. In some embodiments, the second chimeric receptor comprises a hinge region (e.g., a CD8α hinge region) located between the C-terminus of the second extracellular domain and the N-terminus of the second transmembrane domain. Including further. In some embodiments, the second chimeric receptor further comprises a signal peptide (eg, CD8α signal peptide) located at the N-terminus of the polypeptide chain. In some embodiments, the second chimeric receptor comprises a polypeptide that includes, from N-terminus to C-terminus, an IL-3 domain, a CD8α hinge region, and a CD8α transmembrane domain. An exemplary second chimeric receptor is shown in FIGS. 1E and 1F.

In some embodiments, the second chimeric receptor comprises (a) a second extracellular domain comprising a second antigen-binding domain, (b) a second transmembrane domain, and (c) a second Contains a polypeptide chain that includes an intracellular signaling domain. In some embodiments, the second extracellular domain further comprises an NKG2D domain, e.g., the second extracellular domain comprises, from the N-terminus to the C-terminus, an NKG2D domain and a second antigen-binding domain, or Contains a second antigen binding domain and an NKG2D domain. In some embodiments, the second antigen binding domain is CD19, CD20, CD22, CD33, CD38, BCMA, CS1, ROR1, GPC3, CD123, IL-13R, CD138, c-Met, EGFR, EGFRvIII, HER2 , HER3, GD-2, NY-ESO-1, MAGE A3, and glycolipid F77. In some embodiments, the second antigen binding domain is derived from a molecule selected from the group consisting of NKG2A, NKG2C, NKG2F, IL-3, IL-13, LLT1, AICL, DNAM-1, and NKp80. A ligand or a ligand binding domain. In some embodiments, the second antigen binding domain is a CD123 binding domain, such as an IL-3 domain. In some embodiments, the second transmembrane domain is derived from a molecule selected from the group consisting of CD8α, CD4, CD28, CD137, CD80, CD86, CD152, and PD1. In some embodiments, the second intracellular signaling domain comprises a costimulatory signaling domain. In some embodiments, the costimulatory signaling domain is of CD27, CD28, 4-1BB, OX40, CD30, ICOS, CD40, CD3, LFA-1, CD2, CD7, LIGHT, NKG2C, B7-H3, CD83. A costimulatory molecule selected from the group consisting of a ligand, and a combination thereof. In some embodiments, the second intracellular signaling domain comprises the primary intracellular signaling domain of an immune effector cell (eg, a T cell). In some embodiments, the primary intracellular signaling domain is derived from CD3ζ. In some embodiments, the second intracellular signaling domain does not include the primary intracellular signaling domain. In some embodiments, the second chimeric receptor further comprises a hinge region (eg, a CD8α hinge region) located between the C-terminus of the extracellular domain and the N-terminus of the transmembrane domain. In some embodiments, the second chimeric receptor further comprises a signal peptide (eg, CD8α signal peptide) located at the N-terminus of the polypeptide chain. In some embodiments, the second chimeric receptor comprises, from N-terminus to C-terminus, an IL-3 domain, a CD8α hinge region, a CD8α transmembrane domain, and a costimulatory signaling domain derived from 4-1BB. Contains polypeptide chains.

In some embodiments, a dual chimeric receptor system is provided comprising (i) a first chimeric receptor and (ii) a second chimeric receptor, wherein (i) the first chimeric receptor is one polypeptide chain and a second polypeptide chain, each of the first polypeptide chain and the second polypeptide chain comprising (a) a first extracellular domain comprising an NKG2D domain; (b) (c) a first intracellular signaling domain, and (ii) the second chimeric receptor comprises (a) a second antigen binding domain (e.g., an IL-3 domain). (b) a second transmembrane domain, and optionally (c) a second intracellular signaling domain. In some embodiments, the NKG2D domain of the first polypeptide chain is cross-linked to the NKG2D domain of the second polypeptide chain by one or more disulfide bonds. In some embodiments, the first transmembrane domain and/or the second transmembrane domain are derived from a molecule selected from the group consisting of CD8α, CD4, CD28, CD137, CD80, CD86, CD152, and PD1. do. In some embodiments, the first intracellular signaling domain comprises the primary intracellular signaling domain of an immune effector cell (eg, a T cell). In some embodiments, the primary intracellular signaling domain is derived from CD3ζ. In some embodiments, the first intracellular signaling domain and/or the second intracellular domain comprises a costimulatory signaling domain. In some embodiments, the costimulatory signaling domain is of CD27, CD28, 4-1BB, OX40, CD30, ICOS, CD40, CD3, LFA-1, CD2, CD7, LIGHT, NKG2C, B7-H3, CD83. a costimulatory molecule selected from the group consisting of a ligand, and a combination thereof. In some embodiments, the second chimeric receptor does not include an intracellular signaling domain. In some embodiments, each polypeptide chain further comprises a hinge region (e.g., a CD8α hinge region) located between the C-terminus of the first extracellular domain and the N-terminus of the first transmembrane domain. . In some embodiments, each polypeptide chain further comprises a signal peptide (eg, CD8α signal peptide) located at the N-terminus of each polypeptide chain. In some embodiments, each of the first polypeptide chain and the second polypeptide chain comprises, from N-terminus to C-terminus, a co-nucleotide derived from the NKG2D domain, the CD8α hinge region, the CD8α transmembrane domain, 4-1BB. It contains a stimulatory signaling domain, and a primary intracellular signaling domain derived from CD3ζ. In some embodiments, the third polypeptide chain includes, from the N-terminus to the C-terminus, an IL-3 domain, and a CD8α transmembrane domain. An exemplary dual chimeric receptor system is shown in Figure 1F.

In some embodiments, the first chimeric receptor polypeptide and the second chimeric receptor polypeptide are expressed by a polycistronic nucleic acid construct. For example, a first chimeric receptor polypeptide is fused to a second chimeric receptor polypeptide by a self-cleaving peptide. Exemplary self-cleavable peptides include, but are not limited to, T2A, P2A, and F2A peptides. T2A peptides have been described, eg, Szymczak AL. et al. , Correction of multi-gene deficiency in vivo using a "self-cleaving" 2A peptide-based retroviral vector. See Nat Biotechnol 2004; 22(5) 589-594.

In some embodiments, one or more isolated nucleic acids are provided that encode any one of the dual chimeric receptor systems described herein. In some embodiments, an isolated nucleic acid is provided comprising a first nucleic acid sequence encoding a first chimeric receptor and a second nucleic acid sequence encoding a second chimeric receptor, , the first nucleic acid sequence is operably linked to the second nucleic acid sequence via a third nucleic acid sequence encoding a self-cleavable peptide (eg, T2A). In some embodiments, where the first chimeric receptor is a dimeric chimeric receptor having two identical polypeptide chains, the first nucleic acid is a monomeric subunit of the first chimeric receptor. , ie encodes a single copy of the polypeptide chain.

In some embodiments, about 85%, 86%, 87%, 88%, 89%, 90%, 91%, Provided is a chimeric receptor comprising an amino acid sequence having at least one of 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. be done. In some embodiments, an NKG2DxIL-3 dual chimeric receptor system is provided that includes a first chimeric receptor and a second chimeric receptor, the first chimeric receptor comprising the amino acid of SEQ ID NO: 34. Approximately 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, wherein the second chimeric receptor has about 85%, 86%, 87%, 88% sequence identity to the amino acid sequence of SEQ ID NO: 41. , 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. Contains amino acid sequence. In some embodiments, an NKG2DxIL-3 dual chimeric receptor system is provided that includes a first chimeric receptor and a second chimeric receptor, the first chimeric receptor comprising the amino acid of SEQ ID NO: 35. Approximately 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, wherein the second chimeric receptor has about 85%, 86%, 87%, 88% sequence identity to the amino acid sequence of SEQ ID NO: 42. , 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. Contains amino acid sequence. In some embodiments, about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95% for the amino acid sequence of SEQ ID NO: 36 or 37. %, 96%, 97%, 98%, 99%, or 100% sequence identity. In some embodiments, about 85%, 86%, 87%, 88%, 89%, 90% for a nucleic acid sequence selected from the group consisting of SEQ ID NOS: 26-27, 39-40, and 43-44. %, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. is provided. In some embodiments, the isolated nucleic acid is DNA. In some embodiments, the isolated nucleic acid is RNA (eg, mRNA). In some embodiments, one or more vectors containing any one or more of the dual chimeric receptor systems or chimeric receptor-encoding nucleic acids described above are provided. In some embodiments, the vector is an expression vector. In some embodiments, the vector is a viral vector, such as a lentiviral vector. In some embodiments, the vector is a non-viral vector. An exemplary dual chimeric receptor system is shown below.
Table 2 Exemplary NKG2D x IL-3 dual chimeric receptor system.
extracellular domain

The chimeric receptors described herein, including multispecific chimeric receptors, a first chimeric receptor and a second chimeric receptor of a dual chimeric receptor system, include one or more NKG2D domains and/or or an extracellular domain having one or more (eg, any one of 1, 2, 3, 4, 5, 6, or more) antigen binding domains, including a second antigen binding domain. The NKG2D domain and the second antigen binding domain may be fused directly to each other via a peptide bond, via a peptide linker, or via a dimerization motif such as a leucine or cysteine zipper. good.
NKG2D domain

The extracellular domain of the chimeric receptor includes one or more NKG2D domains. In some embodiments, the extracellular domain of the chimeric receptor comprises a single NKG2D domain. In some embodiments, the extracellular domain includes a first NKG2D domain and a second NKG2D domain. In some embodiments, the first NKG2D domain and the second NKG2D domain are the same. In some embodiments, the first NKG2D domain and the second NKG2D domain are different. In some embodiments, the first NKG2D domain and the second NKG2D domain are fused to each other directly by a peptide bond or via a peptide linker.

In some embodiments, the NKG2D domain is derived from a human NKG2D molecule. In some embodiments, the NKG2D domain is derived from the extracellular domain of NKG2D, such as human NKG2D. In some embodiments, the NKG2D domain is a forward NKG2D domain, ie, a domain that has an amino acid sequence in the same order as a wild-type NKG2D domain. In some embodiments, the NKG2D domain comprises any of at least about 100, 105, 110, 115, 120, 125, 130, 135, 140, 150 or more amino acids from the extracellular domain of wild type NKG2D. or one. In some embodiments, the NKG2D domain is a reverse NKG2D domain, ie, a domain with a sequence in the opposite direction to the wild-type NKG2D domain. In some embodiments, the NKG2D domain (including the first NKG2D domain and/or the second NKG2D domain) is about 85%, 86%, 87%, 88%, 89% relative to SEQ ID NO: 7 or 8. %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. including. In some embodiments, the NKG2D domain comprises at least 1, 2, 3, 4, 5, or more amino acid substitutions (eg, conservative amino acid substitutions) compared to the corresponding wild-type sequence of NKG2D.

NKG2D is a unique member of the NKG2 family of C-type lectin receptors that stimulate or inhibit the cytotoxic activity of NK cells. NKG2D is a type II transmembrane-anchored glycoprotein expressed primarily on the surface of NK cells and CD8 ⁺ T cells (eg, αβ T cells and γδ T cells). NKG2D is highly conserved across multiple species, with 70% sequence identity shared between the human and mouse receptors. Unlike other NKG2 receptors, which form heterodimers with CD94 and bind to non-classical MHC glycoprotein class I, NKG2D forms homodimers and binds to cellular stress-inducing molecules. Accumulating evidence indicates that NKG2D plays a critical role in immune surveillance against stressed or abnormal cells, such as autologous tumor cells and virus-infected cells.

MIC molecules (MHC class I chain-associated proteins A and B, or MICA and MICB) encoded by genes of the MHC family, ULBP molecules (UL16 binding protein, also known as RAET1 protein) clustered on human chromosome 6 ), various NKG2D ligands have been identified in humans (Bahram et al. 2005). All NKG2D ligands are homologous to MHC class I molecules and exhibit considerable allelic diversity. Although NKG2D ligand RNA is widely expressed in all tissues and organs of the body, NKG2D ligand is generally absent from the surface of normal adult cells (Le Bert and Gasser 2014). However, NKG2D ligand expression is induced or upregulated primarily in tissues of epithelial origin in response to cellular stresses, including heat shock, DNA damage, and stalled DNA replication. The presence of NKG2D ligands on cells marks them for targeting and potential elimination by NK cells (Le Bert and Gasser 2014). Interestingly, across a variety of hematological and solid tumors, the high activity of DNA repair pathways in transformed cells results in the expression of NKG2D ligands, thereby rendering these cells susceptible to NK-mediated lysis ( Sentman et al. 2006).

NKG2D is encoded by the KLRK1 gene. NKG2D contains three domains: a cytoplasmic domain (residues 1-51 of human NKG2D), a transmembrane domain (residues 52-72 of human NKG2D), and an extracellular domain (residues 73-216 of human NKG2D). , a transmembrane receptor protein. The extracellular domain of NKG2D contains a C-type lectin domain (residues 98-213 of human NKG2D).
second antigen binding domain

The second antigen binding domain specifically binds to a cell surface molecule. The second antigen binding domain may be selected to recognize an antigen that acts as a cell surface marker on target cells associated with a particular disease state. The antigen targeted by the second antigen binding domain may be directly or indirectly involved in the disease. In some embodiments, the antigen is a tumor antigen. In some embodiments, the tumor antigen is associated with a B cell malignancy.

Tumor antigens are proteins produced by tumor cells that can elicit an immune response, particularly a T cell-mediated immune response. The choice of target antigen for the present invention will depend on the particular type of cancer being treated. Exemplary tumor antigens include, for example, glioma-associated antigen, carcinoembryonic antigen (CEA), beta-human chorionic gonadotropin, alpha-fetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1 , MN-CAIX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxylesterase, mut hsp70-2, M-CSF, prostase, prostate-specific antigen (PSA), PAP, NY-ESO-1, LAGE -la, p53, prostein, PSMA, HER2/neu, survivin and telomerase, prostate cancer tumor antigen-1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, ephrinB2, CD22, insulin growth factor (IGF) )-I, IGF-II, IGF-I receptor, and mesothelin.

In some embodiments, the tumor antigen is a tumor-specific antigen (TSA) or a tumor-associated antigen (TAA). TSA is specific to tumor cells and does not occur on other cells in the body. TAA-related antigens are not unique to tumor cells, but rather are also expressed on normal cells under conditions that fail to induce a state of immune tolerance to the antigen. Expression of antigens on tumors may occur under conditions in which the immune system is able to respond to the antigen. TAAs may be antigens expressed on normal cells during fetal development when the immune system is immature and unable to respond, or TAAs are normally present at very low levels on normal cells; It may also be an antigen that is expressed at much higher levels on tumor cells.

Non-limiting examples of TSA or TAA antigens include: differentiation antigens such as MART-1/MelanA (MART-I), gp100 (Pmel17), tyrosinase, TRP-1, TRP-2, and MAGE- 1. Tumor-specific multilineage antigens such as MAGE-3, BAGE, GAGE-1, GAGE-2, pl5; overexpressed embryonic antigens such as CEA; overexpressed oncogenes such as p53, Ras, HER2/neu and Mutated tumor suppressor genes; unique tumor antigens resulting from chromosomal translocations such as BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR; and Epstein-Barr virus antigens EBVA and human papillomavirus (HPV) Viral antigens such as antigens E6 and E7. Other large protein-based antigens include TSP-180, MAGE-4, MAGE-5, MAGE-6, RAGE, NY-ESO, pl85erbB2, pl80erbB-3, c-met, nm-23HI, PSA, TAG- 72, CA19-9, CA72-4, CAM17.1, NuMa, K-ras, beta-catenin, CDK4, Mum-1, p15, p16, 43-9F, 5T4, 791Tgp72, alpha-fetoprotein, beta-HCG, BCA225, BTAA, CA125, CA15-3\CA27.29\BCAA, CA195, CA242, CA-50, CAM43, CD68\P1, CO-029, FGF-5, G250, Ga733\EpCAM, HTgp-175, M344, MA- 50, MG7-Ag, MOV18, NB/70K, NY-CO-1, RCAS1, SDCCAG16, TA-90\Mac-2 binding protein\cyclophilin C-related protein, TAAL6, TAG72, TLP, and TPS.

The second antigen binding domain can be of any suitable type. In some embodiments, the second antigen binding domain is derived from an antibody, such as a four chain antibody, or a single domain antibody, such as a heavy chain only antibody. In some embodiments, the second antigen binding domain is an antibody fragment such as a Fab, Fv, scFv, or VHH. In some embodiments, the second antigen binding domain is CD19, CD20, CD22, CD33, CD38, BCMA, CS1, ROR1, GPC3, CD123, IL-13R, CD138, c-Met, EGFR, EGFRvIII, GD -2, NY-ESO-1, MAGE A3, and glycolipid F77.

In some embodiments, the second antigen binding domain is a ligand binding domain of a ligand or receptor. In some embodiments, the second antigen binding domain is derived from a molecule selected from the group consisting of NKG2A, NKG2C, NKG2F, IL-3, IL-13, LLT1, AICL, DNAM-1, and NKp80. A ligand or a ligand binding domain.
CD123 binding domain

In some embodiments, the second antigen binding domain is a CD123 binding domain. In some embodiments, the CD123 binding domain is an antibody fragment (eg, scFv or VHH) of an anti-CD123 antibody. In some embodiments, the CD123 binding domain is a ligand for CD123 or an IL-3 domain. In some embodiments, the IL-3 domain is derived from human IL-3, such as a full-length or functional fragment of human IL-3. In some embodiments, the CD123 binding domain is about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95% relative to SEQ ID NO:9. %, 96%, 97%, 98%, 99%, or 100% sequence identity.

In some embodiments, chimeric receptors are provided that include (a) an extracellular domain that includes a CD123 binding domain, and (b) a transmembrane domain. In some embodiments, the transmembrane domain is derived from a molecule selected from the group consisting of CD8α, CD4, CD28, CD137, CD80, CD86, CD152, and PD1. In some embodiments, the chimeric receptor further comprises an intracellular signaling domain. In some embodiments, the intracellular signaling domain comprises the primary intracellular signaling domain of an immune effector cell (eg, a T cell). In some embodiments, the primary signaling domain is derived from CD3ζ. In some embodiments, the intracellular signaling domain does not include the primary intracellular signaling domain of the immune effector cell. In some embodiments, the intracellular signaling domain comprises a costimulatory signaling domain. In some embodiments, the intracellular signaling domain consists of (or consists essentially of) one or more costimulatory signaling domains. In some embodiments, the costimulatory signaling domain is of CD27, CD28, 4-1BB, OX40, CD30, ICOS, CD40, CD3, LFA-1, CD2, CD7, LIGHT, NKG2C, B7-H3, CD83. a costimulatory molecule selected from the group consisting of a ligand, and a combination thereof. In some embodiments, the chimeric receptor further comprises a hinge region (eg, a CD8α hinge region) located between the C-terminus of the extracellular domain and the N-terminus of the transmembrane domain. In some embodiments, the chimeric receptor includes, from the N-terminus to the C-terminus, a CD123 binding domain, and a transmembrane domain (CD8α). In some embodiments, the CD123 binding domain is an IL-3 domain. In some embodiments, the CD123 binding domain comprises the amino acid sequence of SEQ ID NO:9.

In some embodiments, about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96 to SEQ ID NO: 41 or 42 %, 97%, 98%, 99%, or 100% sequence identity. In some embodiments, about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96 to SEQ ID NO: 43 or 44. %, 97%, 98%, 99%, or 100% sequence identity.

The IL-3 (interleukin 3) gene is located on chromosome 5 and encodes a protein that is 152 amino acids long. IL-3 is a cytokine that can support a wide range of cellular activities such as cell growth, differentiation, and apoptosis. IL-3 acts by binding to the interleukin 3 receptor (IL-3R), also known as the CD123 antigen. The IL-3R is shared by the receptors for IL-3, colony stimulating factor 2 (CSF2/GM-CSF), and interleukin 5 (IL5), which contain a ligand-specific alpha subunit and a signaling beta subunit. , is a heterodimerization receptor. Activation of IL-3R results in phosphorylation of the βc chain, recruitment of SH2-containing adapter molecules such as Vav1, and downstream signaling through the Jak2/STAT5 and Ras/MAPK pathways.

IL-3R is a 75 kD glycoprotein that becomes 43 kD when hydrolyzed by N-glycosidase. IL-3R has three extracellular domains responsible for specific binding to IL-3, a transmembrane domain, and a short intercellular domain essential for intracellular signal transduction (Sato et al. 1993). IL-3R is a heterodimerizing receptor with low affinity and high specificity for IL-3. When IL-3R binds to IL-3, it is activated and promotes cell proliferation and survival (Liu et al. 2015).

CD123 is overexpressed on AML blasts (ie, myeloblasts). AML blasts and leukemic stem cells (LSCs) in 75-89% of AML patients express CD123. In sharp contrast, on normal hematopoietic stem cells (HSCs), expression of CD123 is low or undetectable (Frankel et al. 2014, Jordan et al. 2000). Apart from AML, CD123 is also overexpressed in various hematological malignancies, including B-lineage acute lymphoblastic leukemia, chronic myeloid leukemia, plasmacytoid dendritic cell neoplasms, and hairy cell leukemia. (Munoz et al. 2001). This expression profile makes CD123 a valuable biomarker in clinical diagnosis, prognosis, and intervention of disease. So far, early-stage clinical trials have demonstrated that CD123-targeted therapy is safe and has no significant adverse effects on hematopoiesis. The antileukemic activity of CD123-targeted therapy in humans is still being investigated.
Dimerization motif

In some embodiments, the extracellular domain includes a dimerization motif and a single NKG2D domain. In some embodiments, the extracellular domain comprises a second antigen binding domain, a single NKG2D domain, and a dimerization motif disposed therebetween. The dimerization motif promotes dimerization of the two polypeptide chains in the chimeric receptor, thereby promoting the formation of NKG2D homodimers and the binding of NKG2D homodimers to NKG2D ligands. Suitable dimerization motifs are known in the art. In some embodiments, the dimerization motif is a leucine zipper. In some embodiments, the leucine zipper comprises the amino acids of SEQ ID NO: 10. In some embodiments, the dimerization motif is a cysteine zipper. Exemplary cysteine zippers are known in the art. For example, Guilaume et al. (2015) PLoS ONE 10(6):e0128779.
peptide linker

In some embodiments, the NKG2D domain and the second antigen binding domain (eg, IL-3 domain) are fused to each other via a peptide linker. In some embodiments, the first NKG2D domain and the second NKG2D domain are fused to each other via a peptide linker. The different domains of a chimeric receptor may also be fused to each other via a peptide linker. The peptide linkers connecting different domains may be the same or different.

Each peptide linker in a chimeric receptor may have the same or different length and/or sequence depending on the structural and/or functional characteristics of the various domains. Each peptide linker may be selected and optimized independently. The length, flexibility, and/or other characteristics of the peptide linker used in the chimeric receptor include, but are not limited to, affinity, specificity, or avidity for one or more particular antigens or epitopes. may have some effect on properties that are not For example, a longer peptide linker may be chosen to ensure that two adjacent domains do not sterically hinder each other. In some embodiments, a short peptide linker may be placed between the transmembrane domain and the intracellular signaling domain of the chimeric receptor. In some embodiments, the peptide linker includes flexible residues (eg, glycine and serine) such that adjacent domains are free to move relative to each other. For example, a glycine-serine pair may be a suitable peptide linker.

Peptide linkers can be any suitable length. In some embodiments, the peptide linker is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, At least one of 20, 25, 30, 35, 40, 50, 75, 100, or longer amino acids in length. In some embodiments, the peptide linker is about 100, 75, 50, 40, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, No more than 8, 7, 6, 5, or shorter amino acids in length. In some embodiments, the length of the peptide linker is from about 1 amino acid to about 10 amino acids, from about 1 amino acid to about 20 amino acids, from about 1 amino acid to about 30 amino acids, from about 5 amino acids to about 15 amino acids, from about 10 amino acids to Any of about 25 amino acids, about 5 amino acids to about 30 amino acids, about 10 amino acids to about 30 amino acids long, about 30 amino acids to about 50 amino acids, about 50 amino acids to about 100 amino acids, or about 1 amino acid to about 100 amino acids It is.

Peptide linkers may have naturally occurring or non-naturally occurring sequences. For example, a sequence derived from the hinge region of a heavy chain only antibody may be used as a linker. See, for example, WO 1996/34103. In some embodiments, the peptide linker is a flexible linker. Exemplary flexible linkers include glycine polymers (G) _n (SEQ ID NO: 28), glycine-serine polymers (including, for example, (GS) _n (SEQ ID NO: 29)), (GSGGS) _n (SEQ ID NO: 30), (GGGS) _n (SEQ ID NO: 31), and (GGGGS) _n (SEQ ID NO: 32), where n is an integer of at least 1), glycine-alanine polymers, alanine-serine polymers, and those known in the art. Other flexible linkers may be mentioned. In some embodiments, the peptide linker comprises an amino acid sequence selected from SEQ ID NOs: 12-15.
transmembrane domain

The multispecific chimeric receptors of the present application, including the first chimeric receptor and the second chimeric receptor of a dual chimeric receptor system, directly or indirectly bind to an extracellular antigen binding domain. Contains transmembrane domains that can be fused. Transmembrane domains may be derived either from natural sources or from synthetic sources. As used herein, "transmembrane domain" refers to any protein structure that is thermodynamically stable in a cell membrane, preferably a eukaryotic cell membrane. Transmembrane domains suitable for use in the chimeric receptors described herein may be obtained from naturally occurring proteins. Alternatively, the transmembrane domain can be a synthetic, non-naturally occurring protein segment, such as a hydrophobic protein segment that is thermodynamically stable in the cell membrane.

Transmembrane domains are classified based on the three-dimensional structure of the transmembrane domain. For example, a transmembrane domain may form an alpha helix, a complex of more than one alpha helix, a beta barrel, or any other stable structure capable of spanning the phospholipid bilayer of a cell. Additionally, transmembrane domains may also or alternatively be classified based on the topology of the transmembrane domain, including the number of transmembrane domains that it crosses, and the orientation of the protein. For example, a single-pass membrane protein crosses the cell membrane once, and a multi-pass membrane protein crosses the cell membrane at least twice (e.g., 2, 3, 4, 5, 6, 7, etc.). more times), traverse. Membrane proteins may be defined as type I, type II, or type III, depending on the topology of their termini and membrane-spanning segments relative to the inside and outside of the cell. Type I membrane proteins have a single transmembrane region and are oriented such that the N-terminus of the protein is on the extracellular side of the lipid bilayer of the cell and the C-terminus of the protein is on the cytoplasmic side. Type II membrane proteins also have a single transmembrane region, but are oriented such that the C-terminus of the protein is on the extracellular side of the cell's lipid bilayer and the N-terminus of the protein is on the cytoplasmic side. Type III membrane proteins have multiple transmembrane segments and may be further subclassified based on the number of transmembrane segments and the location of the N- and C-termini.

In some embodiments, the transmembrane domain of the chimeric receptor described herein is derived from a type I single-pass membrane protein. In some embodiments, transmembrane domains from multipass membrane proteins may also be suitable for use in the chimeric receptors described herein. Multipass membrane proteins may include complex (at least two, three, four, five, six, seven, or more) alpha helices, or beta sheet structures. Preferably, the N-terminus and C-terminus of the multipass membrane protein are present on opposite sides of the lipid bilayer, e.g., the N-terminus of the protein is present on the cytoplasmic side of the lipid bilayer, and the C-terminus of the protein is present on the cytoplasmic side of the lipid bilayer. exists on the outside.

In some embodiments, the transmembrane domain of the chimeric receptor is the alpha, beta, or zeta chain of a T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD134, CD134, CD154, CD154, KIRDS2, OX40, CD27, CD27, LFA -1 (CD11A, CD18), ICOS (CD278), 4-1BB (CD137), Git R, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRFl), CD160, CD19, IL-2Rbeta, IL-2Rgamma, IL-7Ra, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD , CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1 (CD226), SLAMF4 ( CD244, 2B4) , CD84, CD96 (Tactile), CEACAM1, CRT AM, Ly9 (CD229), CD160 (BY55), PSGL1, CDIOO (SEMA4D), SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD150, IPO-3 ), The transmembrane domain is selected from the transmembrane domains of BLAME (SLAMF8), SELPLG (CD162), LTBR, PAG/Cbp, NKp44, NKp30, NKp46, NKG2D, and/or NKG2C. In some embodiments, the transmembrane domain is derived from a molecule selected from the group consisting of CD8α, CD4, CD28, 4-1BB, CD80, CD86, CD152, and PD1.

In some embodiments, the transmembrane domain is derived from CD28. In some embodiments, the transmembrane domain is derived from CD8α. In some embodiments, the transmembrane domain is derived from the full-length transmembrane domain of CD8α. In some embodiments, the transmembrane domain is derived from a truncated transmembrane domain of CD8α. In some embodiments, the transmembrane domain is a transmembrane domain of CD8α comprising the amino acid sequence of SEQ ID NO: 4 or 45.

Transmembrane domains for use in the chimeric receptors described herein can also include at least a portion of non-naturally occurring synthetic protein segments. In some embodiments, the transmembrane domain is a non-naturally occurring synthetic alpha helix or beta sheet. In some embodiments, the protein segment is at least approximately 20 amino acids, such as at least 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or longer amino acids. It is. Examples of synthetic transmembrane domains are known in the art, eg, in US Pat. 7,052,906B1 and PCT Publication WO 2000/032776A2, the relevant disclosures of which are incorporated herein by reference.

The transmembrane domain may include a transmembrane region and a cytoplasmic region located C-terminal to the transmembrane domain. The cytoplasmic region of the transmembrane domain may include three or more amino acids and, in some embodiments, helps orient the transmembrane domain in the lipid bilayer. In some embodiments, one or more cysteine residues are present in the transmembrane region of the transmembrane domain. In some embodiments, one or more cysteine residues are present in the cytoplasmic region of the transmembrane domain. In some embodiments, the cytoplasmic region of the transmembrane domain includes positively charged amino acids. In some embodiments, the cytoplasmic region of the transmembrane domain includes the amino acids arginine, serine, and lysine.

In some embodiments, the transmembrane region of the transmembrane domain includes hydrophobic amino acid residues. In some embodiments, the transmembrane domain of the chimeric receptor comprises an artificial hydrophobic sequence. For example, a phenylalanine, tryptophan, and valine triplet may be present at the C-terminus of the transmembrane domain. In some embodiments, the transmembrane region comprises predominantly hydrophobic amino acid residues such as alanine, leucine, isoleucine, methionine, phenylalanine, tryptophan, or valine. In some embodiments, the transmembrane region is hydrophobic. In some embodiments, the transmembrane region comprises a poly-leucine-alanine sequence. The hydropathy, or hydrophilicity index, of a protein or protein segment can be assessed by any method known in the art, such as Kyte and Doolittle hydropathy analysis.
Intracellular signaling domain

The chimeric receptors of this application, including multispecific chimeric receptors, a first chimeric receptor and a second chimeric receptor of a dual chimeric receptor system, include an intracellular signaling domain. The intracellular signaling domain is responsible for activation of at least one of the normal effector functions of the immune effector cell expressing the chimeric receptor. The term "effector function" refers to a specialized function of a cell. The effector function of a T cell may be, for example, cytolytic activity or helper activity, including secretion of cytokines. Accordingly, the term "cytoplasmic signaling domain" refers to the portion of a protein that transmits effector function signals and instructs cells to perform specialized functions. Typically, the entire cytoplasmic signaling domain can be used, but in many cases it is not necessary to use the entire chain. To the extent that truncated portions of the cytoplasmic signaling domain are used, such truncated portions may be used in place of the intact chain so long as they transmit the effector function signal. Accordingly, the term cytoplasmic signaling domain is intended to include any truncated portion of the cytoplasmic signaling domain sufficient to transmit an effector function signal.

In some embodiments, the intracellular signaling domain comprises the primary intracellular signaling domain of an immune effector cell. In some embodiments, the chimeric receptor comprises an intracellular signaling domain consisting essentially of the primary intracellular signaling domain of an immune effector cell. "Primary intracellular signaling domain" refers to cytoplasmic signaling sequences that act in a stimulating manner to elicit immune effector functions. In some embodiments, the primary intracellular signaling domain contains a signaling motif known as an immunoreceptor activation tyrosine motif or ITAM. As used herein, "ITAM" is a conserved protein motif commonly present in the tail of signaling molecules expressed in many immune cells. The motif may include two repeats of the amino acid sequence YxxL/I separated by 6-8 amino acids, where each x is independently any amino acid, and the conserved motif YxxL/Ix (6-8 ) YxxL/I is generated. ITAM within the signaling molecule is important for intracellular signal transduction, which is mediated, at least in part, by phosphorylation of tyrosine residues in ITAM following activation of the signaling molecule. . ITAM may also function as a docking site for other proteins involved in signal transduction pathways. Exemplary ITAM-containing primary cytoplasmic signaling sequences include CD3ζ, FcR gamma (FCER1G), FcR beta (Fc epsilon Rib), CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d. Examples include those derived from.

In some embodiments, the primary intracellular signaling domain is derived from CD3ζ. In some embodiments, the intracellular signaling domain consists of the cytoplasmic signaling domain of CD3ζ. In some embodiments, the primary intracellular signaling domain is the cytoplasmic signaling domain of wild type CD3ζ. In some embodiments, the primary intracellular signaling domain of wild-type CD3ζ comprises the amino acid sequence of SEQ ID NO:6.
costimulatory signaling domain

Many immune effector cells require co-stimulation in addition to stimulation of antigen-specific signals, not only to activate cell effector functions, but also to promote cell proliferation, differentiation, and survival. In some embodiments, the intracellular signaling domain includes at least one costimulatory signaling domain. As used herein, the term "costimulatory signaling domain" refers to at least a portion of a protein that mediates intracellular signal transduction to elicit an immune response, such as effector function. The costimulatory signaling domain of the chimeric receptors described herein can be a cytoplasmic signaling domain derived from a costimulatory protein, where the costimulatory protein transduces a signal and is transmitted to T cells, NK cells, macrophages, and Modulate responses mediated by immune cells such as neutrophils, or eosinophils. A "costimulatory signaling domain" can be the cytoplasmic portion of a costimulatory molecule. The term "costimulatory molecule" refers to a cognate binding partner on an immune cell (e.g., a T cell) that specifically binds a costimulatory ligand, thereby promoting proliferation and survival, including but not limited to mediate costimulatory responses by immune cells, such as

In some embodiments, the intracellular signaling domain comprises a single costimulatory signaling domain. In some embodiments, the intracellular signaling domain comprises two or more (eg, any of about 2, 3, 4, or more) costimulatory signaling domains. In some embodiments, the intracellular signaling domain comprises two or more of the same costimulatory signaling domains, eg, two copies of the costimulatory signaling domain of CD28. In some embodiments, the intracellular signaling domain comprises two or more costimulatory signaling domains from different costimulatory proteins, such as any two or more costimulatory proteins described herein. include. In some embodiments, the intracellular signaling domain comprises a primary intracellular signaling domain (eg, the cytoplasmic signaling domain of CD3ζ) and one or more costimulatory signaling domains. In some embodiments, one or more costimulatory signaling domains and a primary intracellular signaling domain (eg, the cytoplasmic signaling domain of CD3ζ) are fused together via an optional peptide linker. The primary intracellular signaling domain and one or more costimulatory signaling domains may be arranged in any suitable order. In some embodiments, one or more costimulatory signaling domains are located between the transmembrane domain and the primary intracellular signaling domain (eg, the cytoplasmic signaling domain of CD3ζ). Multiple costimulatory signaling domains may provide additional or synergistic stimulatory effects.

Activation of costimulatory signaling domains in host cells (e.g., immune cells) causes the cells to increase or decrease cytokine production and secretion, phagocytic properties, proliferation, differentiation, survival, and/or cytotoxicity. May be induced. The costimulatory signaling domain of any costimulatory molecule may be suitable for use in the chimeric receptors described herein. The type of costimulatory signaling domain depends on the type of immune effector cell in which the effector molecule is expressed (e.g., T cell, NK cell, macrophage, neutrophil, or eosinophil) and the desired immune effector function (e.g., ADCC effect). ) and other factors. Examples of costimulatory signaling domains for use in chimeric receptors include, without limitation, the B7/CD28 family (e.g., B7-1/CD80, B7-2/CD86, B7-H1/PD-L1, B7 -H2, B7-H3, B7-H4, B7-H6, B7-H7, BTLA/CD272, CD28, CTLA-4, Gi24/VISTA/B7-H5, ICOS/CD278, PD-1, PD-L2/B7 - DC, and PDCD6); members of the TNF superfamily (e.g., 4-1BB/TNFSF9/CD137, 4-1BB ligand/TNFSF9, BAFF/BLyS/TNFSF13B, BAFF R/TNFRSF13C, CD27/TNFRSF7, CD27 ligand/TNFSF7, CD30/TNFRSF8, CD30 ligand/TNFSF8, CD40/TNFRSF5, CD40/TNFSF5, CD40 ligand/TNFSF5, DR3/TNFRSF25, GITR/TNFRSF18, GITR ligand/TNFSF18, HVEM/TNFRSF14, LI GHT/TNFSF14, lymphotoxin alpha/TNF beta , OX40/TNFRSF4, OX40 ligand/TNFSF4, RELT/TNFRSF19L, TACI/TNFRSF13B, TL1A/TNFSF15, TNF alpha, and TNF RII/TNFRSF1B); members of the SLAM family (e.g., 2B4/CD244/SLAMF 4, BLAME/SLAMF8, CD2 , CD2F-10/SLAMF9, CD48/SLAMF2, CD58/LFA-3, CD84/SLAMF5, CD229/SLAMF3, CRACC/SLAMF7, NTB-A/SLAMF6, and SLAM/CD150); and CD2, CD7, CD53, CD82/Kai-1, CD90/Thy1, CD96, CD160, CD200, CD300a/LMIR1, HLA class I, HLA-DR, Icarus, Integrin alpha 4/CD49d, Integrin alpha 4 beta 1, Integrin alpha 4 beta 7/LPAM- 1, LAG-3, TCL1A, TCL1B, CRTAM, DAP12, Dectin 1/CLEC7A, DPPIV/CD26, EphB6, TIM-1/KIM-1/HAVCR, TIM-4, TSLP, TSLP R, lymphocyte function-related antigen 1 (LFA-1), and any other costimulatory molecules, such as NKG2C.

In some embodiments, the one or more costimulatory signaling domains include CD27, CD28, 4-1BB (i.e., CD137), ICOS, OX40, CD30, CD40, CD3, lymphocyte function-associated antigen 1 (LFA- 1), a ligand that specifically binds to CD2, CD7, LIGHT, NKG2C, B7-H3, and CD83.

In some embodiments, the intracellular signaling domain in the chimeric receptors of the present application comprises a costimulatory signaling domain derived from CD28. In some embodiments, the intracellular signaling domain comprises the cytoplasmic signaling domain of CD3ζ and the costimulatory signaling domain of CD28. In some embodiments, the intracellular signaling domain in the chimeric receptors of the present application comprises a costimulatory signaling domain derived from 4-1BB (ie, CD137). In some embodiments, the intracellular signaling domain comprises the cytoplasmic signaling domain of CD3ζ and the costimulatory signaling domain of 4-1BB. In some embodiments, the intracellular signaling domain comprises a 4-1BB costimulatory signaling domain comprising the amino acid sequence of SEQ ID NO:5.

In some embodiments, the intracellular signaling domains in the chimeric receptors of the present application include the costimulatory signaling domain of CD28 and the costimulatory signaling domain of 4-1BB. In some embodiments, the intracellular signaling domain includes the cytoplasmic signaling domain of CD3ζ, the costimulatory signaling domain of CD28, and the costimulatory signaling domain of 4-1BB. In some embodiments, the intracellular signaling domain is a polypeptide comprising, from the N-terminus to the C-terminus, the costimulatory signaling domain of CD28, the costimulatory signaling domain of 4-1BB, and the cytoplasmic signaling domain of CD3ζ. Contains peptides.

Further within the scope of this disclosure is any of the costimulatory signaling domains described herein, such that the costimulatory signaling domain is capable of modulating the immune response of an immune cell. It is a variant. In some embodiments, the costimulatory signaling domain has up to 10 (e.g., 1, 2, 3, 4, 5, or 8) compared to its wild-type counterpart. Contains variations in amino acid residues. Such costimulatory signaling domains that include one or more amino acid variations may be referred to as variants. Mutations in amino acid residues in costimulatory signaling domains may increase signal transduction and enhance stimulation of immune responses compared to costimulatory signaling domains that do not contain mutations. Mutations in amino acid residues in a costimulatory signaling domain may result in decreased signal transduction and stimulation of an immune response compared to a costimulatory signaling domain that does not contain the mutation.
hinge area

The multispecific chimeric receptors of the present application, including the first chimeric receptor and the second chimeric receptor of a dual chimeric receptor system, have an extracellular antigen-binding domain and a transmembrane domain. It may also include a hinge region located therein. A hinge region is generally an amino acid segment found between two domains of a protein and may allow flexibility of the protein and movement of one or both of the domains relative to each other. Any amino acid sequence that provides such mobility and movement of the extracellular antigen binding domain relative to the transmembrane domain of the effector molecule can be used.

The hinge region may contain any one of about 10-100 amino acids, such as about 15-75 amino acids, 20-50 amino acids, or 30-60 amino acids. In some embodiments, the hinge region has a length of about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 , 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, or 75 amino acids.

In some embodiments, the hinge region is a naturally occurring protein hinge region. The hinge region of any protein known in the art to contain a hinge region is suitable for use in the chimeric receptors described herein. In some embodiments, the hinge region is at least a portion of a naturally occurring protein hinge region, which confers mobility to the chimeric receptor. In some embodiments, the hinge region is derived from CD8α. In some embodiments, the hinge region contains at least 15 (e.g., 20, 25, 30, 35, or 40) contiguous amino acids of a portion of the hinge region of CD8α, e.g., the hinge region of CD8α. It is a fragment. In some embodiments, the hinge region of CD8α comprises the amino acid sequence of SEQ ID NO:3.

Hinge regions of antibodies such as IgG, IgA, IgM, IgE, or IgD antibodies are also suitable for use in the chimeric receptors described herein. In some embodiments, the hinge region is a hinge region that joins the antibody constant domains CH1 and CH2. In some embodiments, the hinge region is of an antibody and includes the hinge region of an antibody and one or more constant regions of the antibody. In some embodiments, the hinge region comprises the hinge region of an antibody and the CH3 constant region of that antibody. In some embodiments, the hinge region comprises the hinge region of an antibody and the CH2 and CH3 constant regions of the antibody. In some embodiments, the antibody is an IgG, IgA, IgM, IgE, or IgD antibody. In some embodiments, the antibody is an IgG antibody. In some embodiments, the antibody is an IgG1, IgG2, IgG3, or IgG4 antibody. In some embodiments, the hinge region comprises the hinge region and the CH2 and CH3 constant regions of an IgG1 antibody. In some embodiments, the hinge region comprises the hinge region and CH3 constant region of an IgG1 antibody.

Non-naturally occurring peptides may also be used as the hinge region of the chimeric receptors described herein. In some embodiments, the hinge region between the C-terminus of the extracellular ligand-binding domain and the N-terminus of the transmembrane domain of the Fc receptor comprises a (GxS)n linker (where x and n are independently , 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or greater).
signal peptide

The multispecific chimeric receptors of the present application, including the first chimeric receptor and the second chimeric receptor of a dual chimeric receptor system, contain a signal peptide (also known as a signal sequence) that is a polypeptide. may be included at the N-terminus of Generally, a signal peptide is a peptide sequence that targets a polypeptide to a desired site in a cell. In some embodiments, the signal peptide targets the effector molecule to the secretory pathway of the cell and allows for incorporation and anchoring of the effector molecule into the lipid bilayer. Signal peptides, including naturally occurring protein signal sequences, or non-naturally occurring synthetic signal sequences, suitable for use in the chimeric receptors described herein will be apparent to those skilled in the art. In some embodiments, the signal peptide is derived from a molecule selected from the group consisting of CD8α, GM-CSF receptor, IL-3, and IgG1 heavy chain. In some embodiments, the signal peptide is derived from CD8α. In some embodiments, the IL-3 signal peptide comprises the amino acid sequence of SEQ ID NO:1. In some embodiments, the CD8α signal peptide comprises the amino acid sequence of SEQ ID NO:2.
III. modified immune effector cells

Further provided in this application is a host cell (e.g., modified immune effector cells).

Accordingly, in some embodiments, an engineered immune effector cell (e.g. T cells) are provided. In some embodiments, the extracellular domain includes a first NKG2D domain and a second NKG2D domain.

In some embodiments, (a) an extracellular domain comprising an NKG2D domain and a second antigen binding domain (e.g., an IL-3 domain), (b) a transmembrane domain, and (c) an intracellular signaling domain. An engineered immune effector cell (eg, T cell) is provided that comprises a multispecific (eg, bispecific) chimeric receptor having a multispecific (eg, bispecific) chimeric receptor.

In some embodiments, (a) an extracellular domain comprising a first NKG2D domain, a second NKG2D domain, and a CD123 binding domain (e.g., an IL-3 domain), (b) a transmembrane domain, and ( c) An engineered immune effector cell (eg, T cell) is provided that comprises a multispecific (eg, bispecific) chimeric receptor having a polypeptide chain that includes an intracellular signaling domain. In some embodiments, the CD123 binding domain is an anti-CD123 antibody fragment (eg, scFv or VHH). In some embodiments, the CD123 binding domain is an IL-3 domain. In some embodiments, the polypeptide chain, from N-terminus to C-terminus, includes an IL-3 domain, a first peptide linker, a first NKG2D domain, a second peptide linker, a second NKG2D domain, a CD8α hinge. region, the CD8α transmembrane domain, the costimulatory signaling domain derived from 4-1BB, and the primary intracellular signaling domain derived from CD3ζ.

In some embodiments, an engineered immune effector cell (e.g., a T cell) is provided that comprises a multispecific (e.g., bispecific) chimeric receptor having a first polypeptide chain and a second polypeptide chain. each polypeptide chain comprises (a) an extracellular domain comprising an NKG2D domain and a CD123 binding domain (e.g., an IL-3 domain), (b) a transmembrane domain, and (c) an intracellular signaling domain. . In some embodiments, the NKG2D domain of the first polypeptide chain is cross-linked to the NKG2D domain of the second polypeptide chain by one or more disulfide bonds. In some embodiments, each of the first polypeptide chain and the second polypeptide chain, from N-terminus to C-terminus, includes an IL-3 domain, a peptide linker, an NKG2D domain, a CD8α hinge region, a CD8α transmembrane domain. , a costimulatory signaling domain derived from 4-1BB, and a primary intracellular signaling domain derived from CD3ζ. In some embodiments, the extracellular domain further comprises a dimerization motif (eg, a leucine zipper or cysteine zipper) positioned between the NKG2D domain and the CD123 binding domain. In some embodiments, each of the first polypeptide chain and the second polypeptide chain, from N-terminus to C-terminus, includes an IL-3 domain, a leucine zipper, an NKG2D domain, a CD8α hinge region, a CD8α transmembrane domain. , a costimulatory signaling domain derived from 4-1BB, and a primary intracellular signaling domain derived from CD3ζ.

In some embodiments, an engineered immune effector cell (e.g., a T cell) is provided that comprises a dual chimeric receptor system having (i) a first chimeric receptor and (ii) a second chimeric receptor; i) the first chimeric receptor comprises a first polypeptide chain and a second polypeptide chain, each of the first polypeptide chain and the second polypeptide chain comprising (a) an NKG2D domain; (b) a first transmembrane domain, and (c) a first intracellular signaling domain, (ii) the second chimeric receptor comprises (a) a second antigen; a third polypeptide chain comprising a second extracellular domain comprising a binding domain, (b) a second transmembrane domain, and optionally (c) a second intracellular signaling domain. In some embodiments, each of the first polypeptide chain and the second polypeptide chain comprises, from N-terminus to C-terminus, a co-nucleotide derived from the NKG2D domain, the CD8α hinge region, the CD8α transmembrane domain, 4-1BB. It contains a stimulatory signaling domain, and a primary intracellular signaling domain derived from CD3ζ. In some embodiments, the second chimeric receptor comprises, from N-terminus to C-terminus, an IL-3 domain, a CD8α hinge region, a CD8α transmembrane domain, and optionally a costimulatory signal derived from 4-1BB. Includes polypeptides that include a transduction domain.

In some embodiments, an engineered immune effector cell (e.g., a T cell) is provided that comprises a dual chimeric receptor system having (i) a first chimeric receptor and (ii) a second chimeric receptor; i) The first chimeric receptor comprises (a) a first extracellular domain comprising a first NKG2D domain and a second NKG2D domain, (b) a first transmembrane domain, and (c) a first (ii) a second chimeric receptor comprising (a) a second extracellular domain comprising a second antigen-binding domain; (b) a second polypeptide chain comprising an intracellular signaling domain; a second polypeptide chain comprising two transmembrane domains, and optionally (c) a second intracellular signaling domain. In some embodiments, the first chimeric receptor comprises, from the N-terminus to the C-terminus, a first NKG2D domain, a second NKG2D domain, a CD8α hinge region, a CD8α transmembrane domain, a co-nucleotide derived from 4-1BB. Includes polypeptides that include a stimulatory signaling domain, and a primary intracellular signaling domain derived from CD3ζ. In some embodiments, the second chimeric receptor comprises, from N-terminus to C-terminus, an IL-3 domain, a CD8α hinge region, a CD8α transmembrane domain, and optionally a costimulatory signal derived from 4-1BB. Includes polypeptides that include a transduction domain.

In some embodiments, the modified immune effector cells express one or more immunomodulators. In some embodiments, the engineered mammalian cell comprises a heterologous nucleic acid encoding an immunomodulator. In some embodiments, a heterologous nucleic acid encoding an immunomodulator is present in a vector encoding a chimeric receptor, multispecific chimeric receptor, or dual chimeric receptor system described herein. . In some embodiments, the immunomodulator is a modulator of Runx3. In some embodiments, the modulator is a polypeptide, such as a polypeptide derived from Runx3 or MITF. In some embodiments, the modulator is an RNA, such as a miRNA or siRNA. Runx3 may help T cells target and infiltrate solid tumors, which is an important factor in the success of T cell-mediated immunotherapy.
vector

This application provides vectors for cloning and expressing any one of the chimeric receptors, multispecific chimeric receptors, or dual chimeric receptor systems described herein. In some embodiments, the vector is suitable for replication and integration in eukaryotic cells, such as mammalian cells. In some embodiments, the vector is a viral vector. Examples of viral vectors include, but are not limited to, adenovirus vectors, adeno-associated virus vectors, lentivirus vectors, retrovirus vectors, vaccinia vectors, herpes simplex virus vectors, and derivatives thereof. Viral vector technology is well known in the art and is described, for example, by Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York), and other virology and molecular biology handbooks.

A number of virus-based systems have been developed for gene transfer into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. Heterologous nucleic acids can be inserted into vectors and packaged into retroviral particles using techniques known in the art. Recombinant virus can then be isolated and delivered to engineered mammalian cells in vitro or ex vivo. Many retroviral systems are known in the art. In some embodiments, adenoviral vectors are used. Many adenoviral vectors are known in the art. In some embodiments, lentiviral vectors are used. In some embodiments, self-inactivating lentiviral vectors are used. For example, self-inactivated lentiviral vectors carrying chimeric receptors, multispecific chimeric receptors, or dual chimeric receptor systems can be packaged using protocols known in the art. The resulting lentiviral vector can be used to transduce mammalian cells (eg, primary human T cells) using methods known in the art. Vectors derived from retroviruses, such as lentiviruses, are suitable tools for achieving long-term gene transfer, as they allow long-term stable integration of the transgene and its propagation in progeny cells. Lentiviral vectors also have low immunogenicity and can transduce non-proliferating cells.

In some embodiments, the vector comprises any one of the nucleic acids encoding a chimeric receptor, multispecific chimeric receptor, or dual chimeric receptor system described herein. Nucleic acids can be cloned into vectors using any molecular cloning method known in the art, including, for example, using restriction endonuclease sites and one or more selectable markers. In some embodiments, the nucleic acid is operably linked to a promoter. A variety of promoters have been studied for gene expression in mammalian cells, and any of those known in the art may be used in the present invention. Promoters may be broadly classified as constitutive promoters or regulated promoters, such as inducible promoters.

In some embodiments, a nucleic acid encoding a chimeric receptor, multispecific chimeric receptor, or dual chimeric receptor system is operably linked to a constitutive promoter. A constitutive promoter allows a heterologous gene (also called a transgene) to be expressed constitutively in a host cell. Exemplary constitutive promoters contemplated herein include the cytomegalovirus (CMV) promoter, human elongation factor 1 alpha (hEF1α), ubiquitin C promoter (UbiC), phosphoglycerokinase promoter (PGK), These include, but are not limited to, the simian virus 40 early promoter (SV40), and the avian β-actin promoter combined with the CMV early enhancer (CAGG). The efficiency of such constitutive promoters in activating transgene expression has been widely compared in numerous studies. For example, Michael C. Milone et al compared the efficiency of CMV, hEF1α, UbiC, and PGK to activate chimeric receptor expression in primary human T cells and found that the hEF1α promoter not only induced the most transgene expression but also CD4 and concluded that it is optimally maintained in CD8 human T cells (Molecular Therapy, 17(8):1453-1464 (2009)).

In some embodiments, a nucleic acid encoding a chimeric receptor, multispecific chimeric receptor, or dual chimeric receptor system is operably linked to an inducible promoter. Inducible promoters belong to the category of regulated promoters. An inducible promoter is one that is inducible by one or more conditions, such as physical conditions, the microenvironment of the modified immune effector cell, or the physiological state of the modified immune effector cell, an inducer (i.e., an inducer), or a combination thereof. can be done. In some embodiments, the inducing conditions do not induce expression of endogenous genes in the engineered mammalian cell and/or in the subject receiving the pharmaceutical composition. In some embodiments, the inducing conditions are from the group consisting of an inducing agent, irradiation (e.g., ionizing radiation, light), temperature (e.g., heat), redox conditions, tumor environment, and activation state of the engineered mammalian cell. selected.

In some embodiments, the vector also selects cells expressing the chimeric receptor, multispecific chimeric receptor, or dual chimeric receptor system from a population of host cells transfected via the lentiviral vector. Contains a selectable marker gene or reporter gene for Both the selectable marker and the reporter gene may be flanked by appropriate regulatory sequences to enable expression in the host cell. For example, the vector may contain transcription terminators, translation terminators, initiation sequences, and promoters useful for regulating expression of the nucleic acid sequence.
immune effector cells

An "immune effector cell" is an immune cell capable of performing immune effector functions. In some embodiments, the immune effector cells express at least FcγRIII and perform ADCC effector functions. Examples of immune effector cells that mediate ADCC include peripheral blood mononuclear cells (PBMCs), natural killer (NK) cells, monocytes, cytotoxic T cells, neutrophils, and eosinophils.

In some embodiments, the immune effector cell is a T cell. In some embodiments, the T cell is CD4+/CD8-, CD4-/CD8+, CD4+/CD8+, CD4-/CD8-, or a combination thereof. In some embodiments, the T cell expresses a chimeric receptor, a multispecific chimeric receptor, or a dual chimeric receptor system, and upon binding to a target cell, such as a CD20+ or CD19+ tumor cell, the T cell expresses an IL-2 , TFN, and/or TNF. In some embodiments, the CD8+ T cells express a chimeric receptor, a multispecific chimeric receptor, or a dual chimeric receptor system and, upon binding to the target cell, lyse the antigen-specific target cell.

In some embodiments, the immune effector cells are NK cells. In other embodiments, the immune effector cells can be established cell lines, such as NK-92 cells.

In some embodiments, immune effector cells are differentiated from stem cells, such as hematopoietic stem cells, pluripotent stem cells, iPS, or embryonic stem cells.

Modified immune effector cells are prepared by introducing a chimeric receptor, multispecific chimeric receptor, or dual chimeric receptor system into an immune effector cell, such as a T cell. In some embodiments, the chimeric receptor, multispecific chimeric receptor, or dual chimeric receptor system is one of the isolated nucleic acids or vectors described herein. into immune effector cells by transfecting any one of them. In some embodiments, the chimeric receptor, multispecific chimeric receptor, or dual chimeric receptor system inserts the protein into the cell membrane while passing the cell through a microfluidic system such as CELL SQUEEZE®. (see, eg, US Patent Application Publication No. 20140287509).

Methods of introducing vectors or isolated nucleic acids into mammalian cells are known in the art. The described vectors can be transferred to immune effector cells by physical, chemical, or biological methods.

Physical methods for introducing vectors into immune effector cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods of producing cells containing vectors and/or exogenous nucleic acids are well known in the art. For example, Sambrook et al. (2001) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York. In some embodiments, vectors are introduced into cells by electroporation.

Biological methods of introducing vectors into immune effector cells include the use of DNA vectors and RNA vectors. Viral vectors have become the most widely used method for inserting genes into mammalian, eg human, cells.

Chemical means for introducing vectors into immune effector cells include macromolecular complexes, nanocapsules, microspheres, beads, and colloidal dispersion systems such as lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. Can be mentioned. An exemplary colloid system for use as a delivery vehicle in vitro is liposomes (eg, artificial membrane vesicles).

In some embodiments, RNA molecules encoding any of the chimeric receptors, multispecific chimeric receptors, or dual chimeric receptor systems described herein are prepared by conventional methods (e.g., in vitro). (transfer method) and then introduced into immune effector cells by known methods such as mRNA electroporation. For example, Rabinovich et al. , Human Gene Therapy 17:1027-1035.

In some embodiments, transduced or transfected immune effector cells are expanded ex vivo after introduction of the vector or isolated nucleic acid. In some embodiments, the transduced or transfected immune effector cells are present for about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 12 days, or 14 days. In some embodiments, the transduced or transfected immune effector cells are further evaluated or screened to select engineered mammalian cells.

Reporter genes may be used to identify potentially transfected cells and assess the functionality of regulatory sequences. Generally, a reporter gene is a gene that encodes a polypeptide that is not present in or expressed by the recipient organism or tissue, and whose expression is indicated by some readily detectable property, such as enzymatic activity. . Expression of the reporter gene is assayed at an appropriate time after the DNA has been introduced into the recipient cells. Suitable reporter genes may include genes encoding luciferase, beta-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or the green fluorescent protein gene (e.g., Ui-Tei et al. FEBS Letters 479: 79-82 (2000)). Suitable expression systems are well known and may be prepared using known techniques or obtained commercially.

Other methods for confirming the presence of nucleic acids encoding chimeric receptors, multispecific chimeric receptors, or dual chimeric receptor systems in engineered immune effector cells include, for example, Southern blotting, Northern blotting, RT - PCR, and molecular biological assays well known to those skilled in the art, such as PCR; biochemical assays, such as detecting the presence or absence of specific peptides, for example by immunological methods (e.g. ELISA and Western blotting); It will be done.
Source of T cells

Prior to T cell expansion and genetic modification, a source of T cells is obtained from an individual. T cells can be obtained from many sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue at the site of infection, ascites, pleural fluid, splenic tissue, and tumors. In some embodiments, any number of T cell lines available in the art may be used. In some embodiments, T cells can be obtained from a unit of blood taken from a subject using any number of techniques known to those skilled in the art, such as the FICOLL™ separation method. In some embodiments, cells from an individual's circulating blood are obtained by apheresis. Apheresis products typically contain T cells, mononuclear cells, granulocytes, B cells, other nucleated white blood cells, red blood cells, and lymphocytes, including platelets. In some embodiments, cells harvested by apheresis may be washed to remove the plasma fraction and placed in a suitable buffer or medium for the next processing step. In some embodiments, cells are washed with phosphate buffered saline (PBS). In some embodiments, the cleaning solution is devoid of calcium, may be devoid of magnesium, or may be devoid of many, if not all, divalent cations. Furthermore, surprisingly, the initial activation step in the absence of calcium results in a broadening of activation. Those skilled in the art will appreciate that the washing step can be carried out using methods known in the art, such as by using semi-automated "flow-through" centrifugation (e.g., Cobe 2991 cell processor, Baxter CytoMate, or Haemonetics Cell Saver 5) according to the manufacturer's instructions. It will be readily understood that this may be accomplished by known methods. After washing, cells are incubated in a variety of biocompatible buffers, such as, for example, Ca ²⁺ and Mg ²⁺ -free PBS, PlasmaLyte A, or other saline solutions with or without buffers. May be resuspended. Alternatively, undesirable components of the apheresis sample may be removed and the cells resuspended directly in culture medium.

In some embodiments, T cells are isolated from peripheral blood by lysing red blood cells and depleting monocytes, e.g., by centrifugation on a PERCOLL™ gradient or counterflow centrifugal elution. Isolated from lymphocytes. Specific subpopulations of T cells, such as CD3+, CD28+, CD4+, CD8+, CD45RA+, and CD45RO+ T cells, can be further isolated by positive or negative selection techniques. For example, in some embodiments, T cells are provided with anti-CD3/anti-CD28 (i.e., 3× 28) Isolated by incubation with conjugated beads. In some embodiments, the time period is about 30 minutes. In further embodiments, the time period ranges from 30 minutes to 36 hours or longer, and all integer values therebetween. In further embodiments, the time period is at least 1, 2, 3, 4, 5, or 6 hours. In some embodiments, the time period is 10-24 hours. In some embodiments, the incubation time period is 24 hours. For isolation of T cells from leukemia patients, using longer incubation times, such as 24 hours, can increase cell yield. In any situation where T cells are scarce compared to other cell types, such as when tumor-infiltrating lymphocytes (TILs) are isolated from tumor tissue or from immunocompromised individuals, longer incubation times can be used. , T cells may be isolated. Additionally, the use of longer incubation times can increase the efficiency of CD8+ T cell capture. Therefore, by simply shortening or lengthening the time that T cells are allowed to bind to CD3/CD28 beads and/or by increasing or decreasing the bead to T cell ratio ( Subpopulations of T cells may or may not be preferentially selected at culture initiation (as further described herein), or at other time points during treatment. Furthermore, by increasing or decreasing the proportion of anti-CD3 and/or anti-CD28 antibodies on beads or other surfaces, T cell subpopulations can be preferentially targeted at the initiation of culture or at other desired time points. You can choose to or not. Those skilled in the art will recognize that multiple selections can also be used. In some embodiments, it may be desirable to perform the selection procedure and use "unselected" cells in the activation and expansion process. "Unselected" cells may be subjected to additional rounds of selection.

Enrichment of T cell populations by negative selection can be achieved with combinations of antibodies directed against surface markers specific to the negatively selected cells. One method involves sorting and/or selection of cells by negative magnetic immunoadherence or flow cytometry using a mixture of monoclonal antibodies directed against cell surface markers present on the cells to be negatively selected. It is. For example, to enrich for CD4+ cells by negative selection, the monoclonal antibody mixture typically includes antibodies against CD14, CD20, CD11b, CD16, HLA-DR, and CD8. In certain embodiments, it may be desirable to enrich or positively select for regulatory T cells that typically express CD4+, CD25+, CD62Lhi, GITR+, and FoxP3+. Alternatively, in certain embodiments, T regulatory cells are depleted by anti-C25 conjugated beads or other similar selection methods.

For isolation of desired cell populations by positive or negative selection, the concentrations of cells and surfaces (eg, particles such as beads) can be varied. In certain embodiments, it may be desirable to significantly reduce the volume in which beads and cells are mixed together (i.e., increase the concentration of cells) to ensure maximum cell and bead contact. be. For example, in one embodiment, a concentration of 2 billion cells/mL is used. In one embodiment, a concentration of 1 billion cells/mL is used. In further embodiments, greater than 100 million cells/mL are used. In further embodiments, concentrations of 10 million, 15 million, 20 million, 25 million, 30 million, 35 million, 40 million, 45 million, or 50 million cells/mL are used. In yet other embodiments, concentrations of cells from 75 million, 80 million, 85 million, 90 million, 95 million, or 100 million cells/mL are used. In further embodiments, a concentration of 125 million or 150 million cells/mL may be used. Using high concentrations can result in increased cell yield, cell activation, and cell expansion. Furthermore, using high cell concentrations may result in cells that may weakly express the target antigen of interest, such as CD28-negative T cells, or in samples where many tumor cells are present (i.e., leukemia blood, tumor tissue, etc.). ) can be captured more efficiently. Such cell populations may have therapeutic value and would be desirable to obtain. For example, using higher concentrations of cells allows for more efficient selection of CD8+ T cells, which normally have weaker CD28 expression.

In some embodiments, it may be desirable to use lower concentrations of cells. By significantly diluting the mixture of T cells and surfaces (eg, particles such as beads), interactions between particles and cells are minimized. This selects cells that express large amounts of the desired antigen that is bound to the particles. For example, at dilute concentrations, CD4+ T cells express higher levels of CD28 and are captured more efficiently than CD8+ T cells. In some embodiments, the cell concentration used is 5×10 ⁶ /mL. In some embodiments, the concentration used can be from about 1 x 10 ⁵ /mL to 1 x 10 ⁶ /mL, and any integer value therebetween.

In some embodiments, cells may be incubated on a rotator at varying speeds and for varying lengths of time at either 2-10° C. or room temperature.

T cells for stimulation can also be frozen after the washing step. Without wishing to be bound by theory, the freezing and subsequent thawing step provides a more homogeneous product by removing granulocytes, and to some extent monocytes, in the cell population. After a washing step to remove plasma and platelets, the cells may be suspended in a freezing solution. Although many freezing solutions and parameters are known in the art and useful in this context, one method is PBS containing 20% DMSO and 8% human serum albumin, or 10% dextran 40 and 5% Dextrose, 20% human serum albumin, and 7.5% DMSO, or 31.25% Plasmalyte-A, 31.25% Dextrose 5%, 0.45% NaCl, 10% Dextran 40 and 5% Dextrose, 20% Human using a culture medium containing serum albumin, and 7.5% DMSO, or other suitable cell freezing medium containing, for example, Hespan and PlasmaLyteA, and then freezing the cells at a rate of 1°C per minute at -80°C. Freeze at °C and store in the gas phase in a liquid nitrogen storage tank. Other methods of controlled freezing may be used, as well as immediate uncontrolled freezing at -20°C or in liquid nitrogen.

In some embodiments, cryopreserved cells are thawed, washed as described herein, and allowed to stand at room temperature for 1 hour before activation.

Further contemplated in this application is to collect a blood sample or apheresis product from a subject during a period of time before the expanded cells described herein may be required. Thus, the source of cells to be expanded can be harvested at any time point needed and desired cells, such as T cells, may benefit from T cell therapy, such as those described herein. The wax is isolated and frozen for later use in T cell therapy for any number of diseases or conditions. In one embodiment, a blood sample or apheresis is obtained from a generally healthy subject. In certain embodiments, the blood sample or apheresis is taken from a generally healthy subject who is at risk of developing the disease, but has not yet developed the disease, and the cells of interest are collected solely for later use. Separated and frozen. In certain embodiments, T cells may be expanded, frozen, and used later. In certain embodiments, the sample is taken from a patient just after being diagnosed with a particular disease described herein, but before receiving any treatment. In further embodiments, the cells are treated with drugs such as natalizumab, efalizumab, antiviral agents, chemotherapy, radiation, immunosuppressants such as cyclosporine, azathioprine, methotrexate, mycophenolic acid, and FK506, antibodies, or campuses. Any number of related treatments including, but not limited to, treatment with other immunodepleting agents, anti-CD3 antibodies, cytoxan, fludarabine, cyclosporine, FK506, rapamycin, mycophenolic acid, steroids, FR901228, and radiation. Isolated from a blood sample or apheresis from a subject prior to certain treatments. These drugs either inhibit the calcium-dependent phosphatase, calcineurin (cyclosporine and FK506) or inhibit the p70S6 kinase (rapamycin), which is important for growth factor-induced signaling ((Liu et al., Cell 66:807-815, 1991; Henderson et al., Immun 73:316-321, 1991; Bierer et al., Curr. Opin. Immun. 5:763-773, 1993). , T cells isolated for the patient and using either a bone marrow transplant or a stem cell transplant, a chemotherapeutic agent (e.g. fludarabine, external radiation therapy (XRT), cyclophosphamide) or an antibody (e.g. OKT3 or Campus). In another embodiment, the cells are frozen for later use in conjunction (e.g., before, concurrently, or after) with ablation therapy. In another embodiment, the cells are treated with a B-cell ablation agent such as a CD20-responsive agent (e.g., Rituxan). It can be isolated prior to therapy and frozen for later use in post-therapy treatments.

In some embodiments, T cells are obtained from the patient directly after treatment. In this regard, after certain cancer treatments, particularly those with drugs that damage the immune system, the quality of T cells obtained immediately after treatment and during the period when the patient is normally recovering from that treatment can be significantly affected ex vivo. It has been observed that there are cases where the ability to scale is optimal or improved. Similarly, after ex vivo manipulation using the methods described herein, these cells may be in a favorable condition for improved engraftment and expansion in vivo. It is therefore contemplated within the context of the present invention to harvest blood cells, including T cells, dendritic cells or other cells of the hematopoietic system, during this recovery phase. Additionally, in certain embodiments, mobilization (e.g., mobilization with GM-CSF) and pretreatment regimens are used to specifically target conditions in which repopulation, recirculation, regeneration, and/or expansion of particular cell types are favorable. can be produced in the subject during a defined period of time following therapy. Exemplary cell types include T cells, B cells, dendritic cells, and other cells of the immune system.
T cell activation and expansion

Whether before or after genetic modification of T cells with the chimeric receptors, multispecific chimeric receptors, or dual chimeric receptor systems described herein, generally, e.g., U.S. Pat. Specification No. 6,352,694, Specification No. 6,534,055, Specification No. 6,905,680, Specification No. 6,692,964, Specification No. 5,858,358 Specification No. 6,887,466, Specification No. 6,905,681, Specification No. 7,144,575, Specification No. 7,067,318, Specification No. No. 7,172,869, No. 7,232,566, No. 7,175,843, No. 5,883,223, No. 6,905,874 6,797,514, 6,867,041, and U.S. Patent Application Publication No. 20060121005, T cells are activated. and can be expanded.

Generally, T cells can be expanded by contacting a surface that has bound thereto an agent that stimulates CD3/TCR complex associated signals and a ligand that stimulates co-stimulatory molecules on the surface of the T cell. Specifically, T cell populations are activated by contact with anti-CD3 antibodies, or antigen-binding fragments thereof, or anti-CD2 antibodies recruited on surfaces, or by protein kinase C activators (e.g., in conjunction with calcium ionophores). may be stimulated as described herein, such as by contact with bryostatin). For costimulation of accessory molecules on the surface of T cells, ligands that bind accessory molecules are used. For example, a population of T cells can be contacted with anti-CD3 and anti-CD28 antibodies under conditions appropriate to stimulate T cell proliferation. Anti-CD3 and anti-CD28 antibodies to stimulate populations of either CD4+ or CD8+ T cells. Examples of anti-CD28 antibodies include 9.3, B-T3, XR-CD28 (Diaclone, Besancon, France) and other methods known in the art (Berg et al., Transplant Proc. 30(8):3975-3977, 1998; Haanen et al., J. Exp. Med. 190(9): 13191328, 1999; Garland et al., J. Immunol Meth. 227(1-2): 53- 63, 1999).

In some embodiments, the primary and co-stimulatory signals for T cells may be provided by different protocols. For example, the agent providing each signal may be in solution or bound to a surface. When bound to surfaces, the agents may be bound to the same surface (ie, the "cis" form) or to separate surfaces (ie, the "trans" form). Alternatively, one drug may be bound to the surface and the other drug in solution. In one embodiment, the agent that provides the costimulatory signal is bound to the cell surface and the agent that provides the primary activation signal is in solution or bound to the surface. In certain embodiments, both agents can be in solution. In another embodiment, the agent may be soluble, in which case it may be cross-linked to a surface such as a cell expressing an Fc receptor or a binding agent that binds to an antibody or agent. In this regard, with respect to artificial antigen presenting cells (aAPCs) contemplated in the present invention for use in T cell activation and expansion, see, for example, U.S. Pat. Please refer to

In some embodiments, T cells are combined with drug-coated beads, the beads and cells are subsequently separated, and the cells are then cultured. In an alternative embodiment, the drug-coated beads and cells are not separated before culturing, but are cultured together. In further embodiments, beads and cells are first concentrated by application of force, such as magnetic force, resulting in increased ligation of cell surface markers, thereby inducing cell stimulation.

As an example, cell surface proteins may be ligated by contacting T cells with paramagnetic beads (3x28 beads) to which anti-CD3 and anti-CD28 are attached. In one embodiment, cells (e.g., 10 ⁴ -10 ⁹ T cells) and beads (e.g., DYNABEADS® M-450, CD3/CD28T paramagnetic beads at a 1:1 ratio) are incubated in a buffer, Preferably combined in PBS (free of divalent cations such as calcium and magnesium). Again, one skilled in the art will readily understand that any cell concentration may be used. For example, target cells may be few in the sample, comprising only 0.01% of the sample, or the entire sample (ie, 100%) may contain target cells of interest. Therefore, any number of cells is within the context of the present invention. In certain embodiments, it may be desirable to significantly reduce the volume in which particles and cells are mixed together (i.e., increase the concentration of cells) to ensure maximum cell and particle contact. be. For example, in one embodiment, a concentration of about 2 billion cells/ml is used. In another embodiment, greater than 100 million cells/mL are used. In further embodiments, concentrations of 10 million, 15 million, 20 million, 25 million, 30 million, 35 million, 40 million, 45 million, or 50 million cells/mL are used. In yet other embodiments, concentrations of cells from 75 million, 80 million, 85 million, 90 million, 95 million, or 100 million cells/mL are used. In further embodiments, a concentration of 125 million or 150 million cells/mL may be used. Using high concentrations can result in increased cell yield, cell activation, and cell expansion. Additionally, high cell concentrations allow for more efficient capture of cells that may weakly express the target antigen of interest, such as CD28-negative T cells. Such cell populations may have therapeutic value and may be desirable to obtain in certain embodiments. For example, using a high concentration of cells allows for more efficient selection of CD8+ T cells, which normally have weaker CD28 expression.

In some embodiments, the mixture may be cultured for an integer value of several hours (about 3 hours) to about 14 days, or any time therebetween. In another embodiment, the mixture may be cultured for 21 days. In one embodiment of the invention, beads and T cells are cultured together for about 8 days. In another embodiment, beads and T cells are cultured together for 2-3 days. Several cycles of stimulation may also be desirable, such that the T cell culture time may be 60 days or more. Appropriate conditions for T cell culture include factors necessary for proliferation and viability such as serum (e.g. fetal bovine or human serum), interleukin 2 (IL-2), insulin, IFN-γ, IL-4, IL -7, GM-CSF, IL-10, IL-12, IL-15, TGFβ, and TNF-α, or any other additives for cell growth known to those skilled in the art). Suitable media (eg, Minimum Essential Medium, or RPMI Medium 1640, or X-vivo 15 (Lonza)) may be included. Other additives for cell growth include, but are not limited to, surfactants, plasmanates, and reducing agents such as N-acetyl-cysteine and 2-mercaptoethanol. The medium contains RPMI1640, AIM-V, DMEM, MEM, α-MEM, F-12, X-Vivo15, and X-Vivo20, amino acids, sodium pyruvate, and vitamins, and is serum-free or or an Optimizer supplemented with an appropriate amount of serum (or plasma), or a defined set of hormones, and/or cytokines in amounts sufficient for T cell growth and expansion. Antibiotics, such as penicillin and streptomycin, are only included in experimental cultures and not in the culture of cells that are to be injected into the subject. Target cells are maintained under conditions necessary to support growth, eg, at an appropriate temperature (eg, 37° C.) and atmosphere (eg, air plus 5% CO ₂ ). T cells exposed to varying stimulation times may exhibit different properties. For example, a typical blood, or apherised peripheral blood mononuclear cell product, has a larger population of helper T cells (TH, CD4+) than the population of cytotoxic or suppressor T cells (TC, CD8). Although ex vivo expansion of T cells by stimulating CD3 and CD28 receptors produces a population of T cells consisting primarily of TH cells before about day 8 to 9, Afterwards, the T cell population includes a larger and growing population of TC cells. Therefore, depending on the therapeutic goal, it may be advantageous to infuse a subject with a T cell population consisting primarily of TH cells. Similarly, if an antigen-specific subset of TC cells has been isolated, it may be beneficial to expand this subset to a larger extent.

Furthermore, in addition to CD4 and CD8 markers, other phenotypic markers change significantly, but largely reproducibly, during the cell expansion process. Such reproducibility therefore allows for the ability to tailor activated T cell products for specific purposes.
IV. pharmaceutical composition

Further provided in this application are modified immune effector cells comprising any one of the chimeric receptors, multispecific chimeric receptors, or dual chimeric receptor systems described herein. a pharmaceutical composition comprising any one, and a pharmaceutically acceptable carrier. Pharmaceutical compositions include a plurality of modified immune effector cells having a desired degree of purity, with optional pharmaceutically acceptable carriers, excipients, or stabilizers, as described in (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)) can be prepared by mixing in a lyophilized formulation or an aqueous solution.

Acceptable carriers, excipients, or stabilizers include buffers, antioxidants, including ascorbic acid, methionine, vitamin E, and sodium metabisulfite, which are non-toxic to the recipient at the dosages and concentrations employed. ; preservatives, tonicity agents, stabilizers, metal complexes (eg, Zn protein complexes); chelating agents such as EDTA and/or nonionic surfactants.

Buffers are used to control pH in a range that optimizes therapeutic efficacy, especially when stability is pH dependent. Preferably, the buffer is present at a concentration ranging from about 50 mM to about 250 mM. Buffers suitable for use with the present invention include both organic and inorganic acids and their salts. For example, citric acid, phosphoric acid, succinic acid, tartaric acid, fumaric acid, gluconic acid, oxalic acid, lactic acid, acetic acid. Additionally, the buffer may include histidine and a trimethylamine salt such as Tris.

Preservatives are added to retard microbial growth and are typically present in the range of 0.2% to 1.0% (w/v). Preservatives suitable for use with the present invention include octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium halides (e.g., chloride, bromide, iodide), benzethonium chloride; thimerosal, phenol, butyl Alcohol or benzyl alcohol; alkylparabens such as methylparaben or propylparaben; catechol; resorcinol; cyclohexanol, 3-pentanol, and m-cresol.

Tonicity agents, sometimes known as "stabilizers," are present to adjust or maintain the osmotic pressure of the liquid in the composition. When used with large, charged biomolecules such as proteins and antibodies, tonicity agents interact with the charged groups of amino acid side chains, thereby lowering the potential for inter- and intramolecular interactions. It is often referred to as a "stabilizer" because of its ability to The tonicity agent may be present in any amount between 0.1% and 25%, preferably 1 and 5% by weight, taking into account the relative amounts of other materials. Preferred tonicity agents include polyhydric sugar alcohols, preferably trihydric or higher sugar alcohols such as glycerin, erythritol, arabitol, xylitol, sorbitol, and mannitol.

Additional excipients include agents that can serve as one or more of the following: (1) fillers, (2) solubility enhancers, (3) stabilizers, and (4) modifying or container agents. An agent that prevents adhesion to walls. Such excipients include polyhydric sugar alcohols (listed above); for example, alanine, glycine, glutamine, asparagine, histidine, arginine, lysine, ornithine, leucine, 2-phenylalanine, glutamic acid, threonine, etc. Amino acids; sucrose, lactose, lactitol, trehalose, stachyose, mannose, sorbose, xylose, ribose, ribitol, myoinisitose, myoinisitol, galactose, galactitol, glycerol, cyclitols (e.g. inositol), polyethylene glycol organic sugars or sugar alcohols such as; sulfur-containing reducing agents such as urea, glutathione, thioctic acid, sodium thioglycolate, thioglycerol, alpha monothioglycerol, and sodium thiosulfate; human serum albumin, bovine serum albumin, gelatin, or low molecular weight proteins such as other immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; monosaccharides (e.g. xylose, mannose, fructose, glucose); disaccharides (e.g. lactose, maltose, sucrose); trisaccharides such as raffinose; and polysaccharides such as dextrin or dextran.

Nonionic surfactants or surfactants (also known as "wetting agents") are present to protect therapeutic proteins against agitation-induced aggregation as well as to aid in solubilization of therapeutic agents. , which also allows the formulation to be exposed to shear surface stress without causing denaturation of the active therapeutic protein or antibody. The nonionic surfactant is present in a range of about 0.05 mg/mL to about 1.0 mg/mL, preferably about 0.07 mg/mL to about 0.2 mg/mL.

Suitable nonionic surfactants include polysorbates (20, 40, 60, 65, 80, etc.), polyoxamers (184, 188, etc.), Pluronic® polyols, Triton®, poly Oxyethylene sorbitan monoether (Tween®-20, Tween®-80, etc.), lauromacrogol 400, polyoxyl stearate 40, polyoxyethylene hydrogenated castor oil 10, 50 and 60, monostearic acid Glycerol, sucrose fatty acid esters, methylcellulose, and carboxymethylcellulose are mentioned. Anionic surfactants that may be used include sodium lauryl sulfate, dioctyl sodium sulfosuccinate, and dioctyl sodium sulfonate. Cationic surfactants include benzalkonium chloride or benzethonium chloride.

Pharmaceutical compositions must be sterile for use in in vivo administration. Pharmaceutical compositions may be rendered sterile by filtering through a sterile filtration membrane. The pharmaceutical compositions herein generally are placed in a container with a sterile access port, such as an intravenous solution bag or a vial with a stopper pierceable by a hypodermic needle.

The route of administration may be in any suitable manner (e.g., by injection or infusion by subcutaneous, intravenous, intraperitoneal, intramuscular, intraarterial, intralesional, or intraarticular routes, local administration, inhalation, or by slow or sustained release means). and according to known and accepted methods, such as by single or multiple bolus administrations or by infusion over an extended period of time.

The pharmaceutical compositions described herein also contain more than one active compound or agent, preferably complementary activities that do not adversely affect each other, as necessary for the particular indication being treated. It may contain those that have. Alternatively, or in addition, the composition may include a cytotoxic agent, chemotherapeutic agent, cytokine, immunosuppressive agent, or growth inhibitory agent. Such molecules are suitably present in combination in an amount effective for the intended purpose.
V. cancer treatment

The application further relates to methods and compositions for use in cellular immunotherapy. In some embodiments, cellular immunotherapy is for treating cancer, including but not limited to hematologic tumors and solid tumors. Any of the chimeric receptors, multispecific chimeric receptors, dual chimeric receptor systems, and modified immune effector cells (e.g., engineered T cells) described herein can be used to treat cancer. may be used in

In some embodiments, the method comprises administering to the individual an effective amount of a pharmaceutical composition for treating cancer (e.g., multiple myeloma, acute lymphoblastic leukemia, or chronic lymphocytosis) in an individual (e.g., a human individual). (1) a chimeric receptor having (a) an extracellular domain comprising an NKG2D domain, (b) a transmembrane domain, and (c) an intracellular signaling domain. (2) a pharmaceutically acceptable carrier. In some embodiments, the chimeric receptor comprises, from N-terminus to C-terminus, the first NKG2D domain, the second NKG2D domain, the CD8α hinge region, the CD8α transmembrane domain, the costimulatory signaling derived from 4-1BB. domain, and a primary intracellular signaling domain derived from CD3ζ.

In some embodiments, the method comprises administering to the individual an effective amount of a pharmaceutical composition for treating cancer (e.g., multiple myeloma, acute lymphoblastic leukemia, or chronic lymphocytosis) in an individual (e.g., a human individual). (1) (a) a first NKG2D domain, a second NKG2D domain, and a CD123 binding domain (e.g., an IL-3 domain); an engineered immune effector cell (e.g., a T cell) comprising a multispecific chimeric receptor having a polypeptide chain comprising an ectodomain, (b) a transmembrane domain, and (c) an intracellular signaling domain; and (2) a medicament. containing a commercially acceptable carrier. In some embodiments, the CD123 binding domain is an anti-CD123 antibody fragment (eg, scFv or VHH). In some embodiments, the CD123 binding domain is an IL-3 domain. In some embodiments, the polypeptide chain, from N-terminus to C-terminus, includes an IL-3 domain, a first peptide linker, a first NKG2D domain, a second peptide linker, a second NKG2D domain, a CD8α hinge. region, the CD8α transmembrane domain, the costimulatory signaling domain derived from 4-1BB, and the primary intracellular signaling domain derived from CD3ζ.

In some embodiments, the method comprises administering to the individual an effective amount of a pharmaceutical composition for treating cancer (e.g., multiple myeloma, acute lymphoblastic leukemia, or chronic lymphocytosis) in an individual (e.g., a human individual). (1) a modified immune effector cell comprising a multispecific chimeric receptor having a first polypeptide chain and a second polypeptide chain (e.g. T cells), each polypeptide chain comprising (a) an extracellular domain comprising an NKG2D domain and a CD123 binding domain (e.g., an IL-3 domain), (b) a transmembrane domain, and (c) an intracellular domain. (2) a pharmaceutically acceptable carrier. In some embodiments, the NKG2D domain of the first polypeptide chain is cross-linked to the NKG2D domain of the second polypeptide chain by one or more disulfide bonds. In some embodiments, each of the first polypeptide chain and the second polypeptide chain, from N-terminus to C-terminus, includes an IL-3 domain, a peptide linker, an NKG2D domain, a CD8α hinge region, a CD8α transmembrane domain. , a costimulatory signaling domain derived from 4-1BB, and a primary intracellular signaling domain derived from CD3ζ. In some embodiments, the extracellular domain further comprises a dimerization motif (eg, a leucine zipper or cysteine zipper) positioned between the NKG2D domain and the CD123 binding domain. In some embodiments, each of the first polypeptide chain and the second polypeptide chain, from N-terminus to C-terminus, includes an IL-3 domain, a leucine zipper, an NKG2D domain, a CD8α hinge region, a CD8α transmembrane domain. , a costimulatory signaling domain derived from 4-1BB, and a primary intracellular signaling domain derived from CD3ζ.

In some embodiments, the method comprises administering to the individual an effective amount of a pharmaceutical composition for treating cancer (e.g., multiple myeloma, acute lymphoblastic leukemia, or chronic lymphocytosis) in an individual (e.g., a human individual). (1) a modified immune system comprising a dual chimeric receptor system having (i) a first chimeric receptor and (ii) a second chimeric receptor. The effector cell (e.g., T cell), the first chimeric receptor comprising a first polypeptide chain and a second polypeptide chain, the first polypeptide chain and the second polypeptide chain each comprising (a) a first extracellular domain comprising an NKG2D domain, (b) a first transmembrane domain, and (c) a first intracellular signaling domain, and (ii) a second chimeric receptor. comprises (a) a second extracellular domain comprising a second antigen binding domain, (b) a second transmembrane domain, and optionally (c) a third intracellular signaling domain comprising (2) a pharmaceutically acceptable carrier. In some embodiments, each of the first polypeptide chain and the second polypeptide chain comprises, from N-terminus to C-terminus, a co-nucleotide derived from the NKG2D domain, the CD8α hinge region, the CD8α transmembrane domain, 4-1BB. It contains a stimulatory signaling domain, and a primary intracellular signaling domain derived from CD3ζ. In some embodiments, the second chimeric receptor comprises, from N-terminus to C-terminus, an IL-3 domain, a CD8α hinge region, a CD8α transmembrane domain, and optionally a costimulatory signal derived from 4-1BB. Contains a polypeptide chain that includes a transduction domain.

The methods described herein are suitable for treating a variety of cancers, including both solid and liquid cancers. In some embodiments, the cancer is multiple myeloma, acute lymphoblastic leukemia, or chronic lymphocytic leukemia. In some embodiments, the cancer is a refractory or relapsed cancer. The methods described herein can be used as a first treatment, a second treatment, a third treatment, or, for example, chemotherapy, surgery, radiation, gene therapy, immunotherapy, bone marrow transplantation. , in an adjuvant or neoadjuvant setting, as a combination therapy with other types of cancer therapy known in the art, such as stem cell transplantation, targeted therapy, cryotherapy, ultrasound therapy, photodynamic therapy, radiofrequency ablation, etc. It may be used in.

Administration of the pharmaceutical composition may be carried out in any convenient manner, such as by injection, infusion, infusion, or implantation. The composition may be administered to a patient intraarterially, subcutaneously, intradermally, intratumorally, intranodally, intramedullarily, intramuscularly, intravenously, or intraperitoneally. In some embodiments, the pharmaceutical composition is administered systemically. In some embodiments, the pharmaceutical composition is administered to an individual by infusion, such as an intravenous infusion. Infusion techniques for immunotherapy are known in the art (see, eg, Rosenberg et al., New Eng. J. of Med. 319: 1676 (1988)). In some embodiments, the composition is administered by intravenous injection.

The dosage and desired drug concentration of the pharmaceutical compositions of the invention may vary depending on the particular use envisaged. Determination of the appropriate dosage or route of administration is well within the skill of those in the art. Animal studies provide reliable guidance for determining effective doses for human treatment. Interspecies adjustment of effective amounts is described by Mordenti, J.; and Chappell, W. "The Use of Interspecies Scaling in Toxicokinetics," In Toxicokinetics and New Drug Development, Yacobi et al. , Eds, Pergamon Press, New York 1989, pp. 42-46. Different formulations will be effective for different treatments and different diseases, and administration intended to treat one organ or tissue may require a different approach than administration to another organ or tissue. It is within the scope of this application that delivery may be necessary.

In some embodiments, the amount of the pharmaceutical composition is effective to produce an objective clinical response in the individual. In some embodiments, the amount of the pharmaceutical composition is effective to produce disease remission (partial or complete) in the individual. In some embodiments, the amount of the pharmaceutical composition is effective to prevent cancer recurrence or disease progression in the individual. In some embodiments, the amount of the pharmaceutical composition is effective to prolong survival (eg, disease-free survival) of an individual. In some embodiments, the pharmaceutical composition is effective in improving the quality of life of an individual.

In some embodiments, the amount of the pharmaceutical composition is effective to inhibit the growth or reduce the size of solid or lymphoid tumors. In some embodiments, the size of the solid tumor or lymphoid tumor is at least about 10% (e.g., about 20%, 30%, 40%, 60%, 70%, 80%, 90%, or 100% (including at least one of them).

In some embodiments, the amount of the pharmaceutical composition is effective to inhibit tumor metastasis in an individual. In some embodiments, at least about 10% (e.g., including at least one of about 20%, 30%, 40%, 60%, 70%, 80%, 90%, or 100%) metastasis is inhibited. In some embodiments, methods of inhibiting lymph node metastasis are provided. In some embodiments, methods of inhibiting metastasis to the lungs are provided. Metastases can be assessed by any method known in the art, such as by blood tests, bone scans, X-ray scans, CT scans, PET scans, and biopsies.
VI. Kits and manufactured articles

Further provided are kits, unit dosages, comprising any of the chimeric receptors, multispecific chimeric receptors, dual chimeric receptor systems, or engineered immune effector cells described herein. and manufactured articles. In some embodiments, a kit is provided that contains any one of the pharmaceutical compositions described herein and preferably provides instructions for its use.

The kits of the present application are in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging (eg, sealed mylar or plastic bags), and the like. Kits optionally provide additional components such as buffers and instructional information. Accordingly, the present application also provides articles of manufacture, including vials (eg, sealed vials), bottles, jars, flexible packages, and the like.

The article of manufacture may include a container and a label or package insert affixed to or associated with the container. Suitable containers include, for example, bottles, vials, syringes, and the like. The container may be formed from a variety of materials, such as glass or synthetic resin. Generally, the container holds a composition effective to treat a disease or disease described herein (e.g., cancer) and may have a sterile access port (e.g., the container can It may be an infusion solution bag or a vial with a stopper pierceable by a hypodermic needle). The label or package insert indicates that the composition is used to treat a particular condition in an individual. The label or package insert further includes instructions for administering the composition to an individual. The label may indicate instructions for reconstitution and/or use. The container holding the pharmaceutical composition may be a multi-use vial, which allows for repeated administration (eg, 2 to 6 doses) of the reconstituted formulation. Package insert refers to the instructions customarily included in commercial packaging for therapeutic products, which instructions contain the indications, usage, dosage, administration, contraindications, and/or warnings regarding the use of such therapeutic products. Contains information about. Additionally, the article of manufacture may further include a second container containing a pharmaceutically acceptable buffer such as bacteriostatic water for injection (BWFI), phosphate buffered saline, Ringer's solution, and dextrose solution. The article of manufacture may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

The kit or article of manufacture may include a plurality of unit doses of the pharmaceutical composition and instructions for use packaged in quantities sufficient for storage and use in pharmacies (eg, hospitals, pharmacies, and compounding pharmacies).

The following examples are intended to be purely illustrative of the present application and therefore should not be considered as limiting the invention in any way. The following examples and detailed description are presented by way of illustration and not as a limitation.
Example 1 Preparation of NKG2D x IL-3 chimeric receptor and dual NKG2D/IL-3 chimeric receptor system

This example describes the design and preparation of exemplary bispecific chimeric receptors, bichimeric receptor systems, and engineered T cells. Design of bispecific chimeric receptors and bichimeric receptor system constructs

Tables 1 and 2 show the components of the seven constructs and the corresponding sequences.

Construct 1: Monomeric NKG2D x IL-3 chimeric receptor (LIC2001). This bispecific chimeric receptor comprises a single polypeptide chain having, from N-terminus to C-terminus, an extracellular domain, a transmembrane domain, and an intracellular signaling domain, where the extracellular domain is From the N-terminus to the C-terminus, an IL-3 domain specific for CD123 binding, a first peptide linker, a first NKG2D domain with an engineered arginine residue at its N-terminus, a second peptide linker, and It contains a second NKG2D domain with an engineered aspartate residue at its C-terminus. The NKG2D domain is responsible for NKG2D ligand binding when dimerized. The engineered arginine and aspartate residues may associate with each other to prevent the NKG2D dimer from binding to free NKG2D ligand that is not bound to tumor cells. A nucleic acid construct encodes a single polypeptide chain.

Construct 2: Monomeric NKG2D x IL-3 chimeric receptor (LIC2001-1). This bispecific chimeric receptor comprises a single polypeptide chain having, from N-terminus to C-terminus, an extracellular domain, a transmembrane domain, and an intracellular signaling domain, where the extracellular domain is From the N-terminus to the C-terminus, it includes an IL-3 domain specific for CD123 binding, a first peptide linker, a first NKG2D domain, a second peptide linker, and a second NKG2D domain. The NKG2D domain is responsible for NKG2D ligand binding when dimerized. A nucleic acid construct encodes a single polypeptide chain. For comparison, an extracellular domain comprising a first forward NKG2D domain, a peptide linker, and a second forward NKG2D domain; a transmembrane domain (CD8α), and an intracellular signaling domain (4-1BB and CD3ζ). A monospecific NKG2D chimeric receptor (LIC2001-2) containing from the N-terminus to the C-terminus was constructed. The amino acid sequence of LIC2001-2 is SEQ ID NO: 33, and the nucleic acid sequence of LIC2001-2 is SEQ ID NO: 38.

Construct 3: Dimeric NKG2D x IL-3 chimeric receptor with leucine zipper motif (LIC2002). This bispecific chimeric receptor comprises two identical polypeptide chains, each having an extracellular domain, a transmembrane domain, and an intracellular signaling domain from the N-terminus to the C-terminus. , where the extracellular domain contains, from the N-terminus to the C-terminus, an IL-3 domain specific for CD123 binding, a leucine zipper motif, and an inverted NKG2D domain responsible for NKG2D ligand binding upon homodimerization. include. A nucleic acid construct encodes a single copy of a polypeptide chain.

Construct 4: Dimeric NKG2D x IL-3 chimeric receptor without leucine zipper motif (LIC2002-1). This bispecific chimeric receptor comprises two identical polypeptide chains, each having an extracellular domain, a transmembrane domain, and an intracellular signaling domain from the N-terminus to the C-terminus. , where the extracellular domain comprises, from N-terminus to C-terminus, an IL-3 domain specific for CD123 binding, a peptide linker, and an inverted NKG2D domain responsible for NKG2D ligand binding upon homodimerization. . A nucleic acid construct encodes a single copy of a polypeptide chain.

Construct 5: Dimeric NKG2D x IL-3 chimeric receptor with leucine zipper motif (LIC2002-2). This bispecific chimeric receptor comprises two identical polypeptide chains, each polypeptide chain having, from the N-terminus to the C-terminus, an extracellular domain, a transmembrane domain, and an intracellular signaling domain. , where the extracellular domain contains, from the N-terminus to the C-terminus, an IL-3 domain specific for CD123 binding, a leucine zipper motif, and a forward NKG2D domain responsible for NKG2D ligand binding upon homodimerization. include. A nucleic acid construct encodes a single copy of a polypeptide chain.

Construct 6: Dual NKG2D/IL-3 chimeric receptor system (LIC2003). A polycistronic nucleic acid construct for a dual NKG2D/IL-3 chimeric receptor system is designed. The construct encodes for a first polypeptide and a second polypeptide, the first polypeptide having a reverse NKG2D structure from the N-terminus to the C-terminus that is responsible for NKG2D ligand binding upon homodimerization. and a forward NKG2D domain, an extracellular domain, a transmembrane domain, and an intracellular signaling domain, followed by a T2A self-cleavable peptide, and the second polypeptide, from the N-terminus to the C-terminus, It contains a specific IL-3 domain followed by a transmembrane domain without an intracellular signaling domain. When the construct is expressed, the first polypeptide forms a first chimeric receptor and the second polypeptide forms a second chimeric receptor.

Construct 7: Dual NKG2D/IL-3 chimeric receptor system (LIC2004). A polycistronic nucleic acid construct for a dual NKG2D/IL-3 chimeric receptor system is designed. The construct encodes for a first polypeptide and a second polypeptide, wherein the first polypeptide is a forward NKG2D protein that, upon homodimerization, is responsible for NKG2D ligand binding from the N-terminus to the C-terminus. a second polypeptide comprising an extracellular domain, a transmembrane domain, and an intracellular signaling domain, followed by a T2A self-cleavable peptide; domain followed by a transmembrane domain without an intracellular signaling domain. When the construct is expressed, the two copies of the first polypeptide form a dimeric first chimeric receptor and the second polypeptide forms a second chimeric receptor. Monospecific NKG2D chimera containing, from N-terminus to C-terminus, a forward NKG2D domain, an extracellular domain including a CD8α hinge domain; a transmembrane domain (CD8α), and an intracellular signaling domain (4-1BB and CD3ζ) A receptor (LIC2004-1) was constructed. The amino acid sequence of LIC2004-1 is SEQ ID NO: 35, and the nucleic acid sequence of LIC2004-1 is SEQ ID NO: 40. Generation of lentiviral expression vectors

Briefly, pLVX-Puro (Clontech #632164) was used to modify the lentiviral vector by replacing the original promoter with the human elongation factor 1α promoter (hEF1α) gene using EcoRI and XbaI by GenScript. did. The NKG2D×IL-3 chimeric receptor gene or the NKG2D/IL-3 dual chimeric receptor system gene was constructed by GenScript and cloned into the vector by EcoRI/HpaI to obtain the recombinant lentivirus expression plasmid, and the recombinant lentivirus The expression plasmid was further subjected to lentiviral packaging procedure.

pMDLg/pRRE (Addgene #12251), pRSV-Rev (Addgene #12253), and pMD2. A lentiviral packaging plasmid mixture containing G (Addgene #12259) was added to pLVX-NKG2D x IL-3 Chimeric Receptor/Dual Chimeric Receptor System - Puro Premixed with expression plasmid, then mixed properly and incubated for 5 minutes at room temperature. The transfection mixture was then added dropwise to the 293FT cells and mixed gently. Cells were then incubated overnight at 37°C in a 5% _CO2 cell incubator. After centrifugation at 500 g for 10 min at 4°C, the supernatant was collected. After filtering the supernatant through a 0.45 μm PES filter, the viral supernatant was concentrated by 20% sucrose gradient ultracentrifugation. After centrifugation, the supernatant was carefully discarded and the virus pellet was carefully rinsed with pre-chilled DPBS. The concentration of virus was then measured. Virus was appropriately aliquoted and then immediately stored at -80°C. Virus titer was determined by p24 based on the HTRF kit developed by GenScript. The following recombinant lentiviral expression plasmids were prepared corresponding to each of the seven constructs described above: pLLV-LIC2001, pLLV-LIC2001-1, pLLV-LIC2002, pLLV-LIC2002-1, pLLV-LIC2002-2, pLLV -LIC2003, and pLLV-LIC2004, and pLLV-LIC2001-2 and pLLV-LIC2004-1. PBMC preparation

Leukocytes were collected and the cell concentration was adjusted to 5x10 ⁶ cells/mL in R10 medium. The leukocytes were then mixed with 0.9% NaCl solution in a 1:1 (v/v) ratio. 3 mL of lymphoprep medium was added to a 15 mL centrifuge tube and 6 mL of the diluted lymphocyte mixture was slowly layered on top of the lymphoprep. The lymphocyte mixture was centrifuged at 800g for 30 minutes at 20°C without brake. The buffy coat of lymphocytes was then collected with a 200 μL pipette. The collected fractions were diluted at least 6 times with 0.9% NaCl or R10 to reduce the density of the solution. The collected fractions were then centrifuged at 250g for 10 minutes at 20°C. The supernatant was aspirated completely and 10 mL of R10 was added to the cell pellet. The mixture was further centrifuged at 250g for 10 minutes at 20°C. The supernatant was then aspirated. 2 mL of R10 containing 100 IU/mL of IL-2, pre-warmed to 37° C., was added to the cell pellet and the cell pellet was gently resuspended. Cell numbers were then counted and PBMC samples were prepared for later experiments. T cell purification

Human T cells were purified from PBMC using the Miltenyi Pan T cell isolation kit (Cat #130-096-535) according to the protocol provided by the manufacturer as follows. Cell number was first determined. The cell suspension was centrifuged at 300g for 10 minutes. The supernatant was then aspirated completely and the cell pellet was resuspended in 40 μL of buffer per 10 ⁷ total cells. 10 μL of Pan TCell Biotin-Antibody Cocktail per 10 ⁷ total cells was added, mixed thoroughly, and incubated in the refrigerator (2-8° C.) for approximately 5 minutes. Then, 30 μL of buffer was added per 10 ⁷ cells. 20 μL of Pan TCell MicroBead Cocktail was added per 10 ⁷ cells. The mixture was mixed well and incubated in the refrigerator (2-8°C) for an additional 10 minutes. A minimum of 500 μL was required for magnetic separation. The LS column was placed in the magnetic field of a suitable MACS separator. The column was prepared by rinsing with 3 mL of buffer. The cell suspension was then applied onto the column and the flow-through containing unlabeled cells representing the enriched T cell fraction was collected. T cells were then collected by washing the column with 3 mL of buffer, and the pass-through unlabeled cells corresponding to the enriched T cells were collected and combined with the flow-through from the previous step. T cells were then resuspended in R10+100 IU/mL IL-2. Primary T cells were then preactivated for 3 days prior to transduction using the humanTCellActivation/ExpansionKit (Miltenyi #130-091-441).

Purified T cells were transfected with each of the recombinant lentiviral vectors or with mRNA encoding each of the chimeric receptor constructs by electroporation and then incubated with 5% _CO2 at 37°C. It was incubated overnight in an incubator. Engineered T cells expressing each of the seven constructs described in this example were prepared.
Expression of chimeric receptors on engineered T cells

mRNA molecules encoding the LIC2002-2 and LIC2004 chimeric receptor constructs, respectively, were delivered to T cells by electroporation. Expression of chimeric receptors was detected using a flow cytometry assay. Briefly, electroporated T cells were collected and rinsed with DPBS, then 2 μL of PE-conjugated CD314 protein (MILTENYI BIOTEC, 130-111-645) or IL-3 for NKG2D domain detection. It was resuspended in 100 μL of DPBS containing 10 μL of PE-conjugated anti-IL-3 antibody (MILTENYI BIOTEC, 130-096-084) for domain detection. The reaction mixture was incubated for 20 minutes at 4°C. Cells were subsequently washed with 200 μL of DPBS, resuspended in DPBS, and analyzed by flow cytometry. As shown in Figure 2, engineered T cells expressed a chimeric receptor with both NKG2D and IL-3 domains.
In vitro cytotoxicity assay

Engineered T cells were collected and seeded into 384-well reaction plates. Target cells were human chronic myeloid leukemia (CML) cell lines K562-Luc and K562-CD123Luc (recombinantly expressing CD123). All cell lines were autologously engineered to express firefly luciferase. To assay the cytotoxicity of engineered T cells towards tumor cells, engineered T cells were engineered for 20 hours at a ratio of effector (engineered T cells) to target cells (“E:T ratio”) of 20:1. T cells were co-incubated with target cells. ONE-GLO™ Luminescent Luciferase Assay Reagent (Promega #E6110) was prepared according to the manufacturer's protocol and added to the co-cultured cells to detect residual luciferase activity in each well. Because luciferase was expressed only in target cells, residual luciferase activity in a well was directly correlated to the number of viable target cells in the well. Maximum luciferase activity was obtained by adding culture medium to target cells in the absence of effector cells. Minimal luciferase activity was determined by adding Triton X-100 at a final concentration of 1% as a positive control. Specific lysis/cytotoxicity was calculated according to the formula: % specific lysis/cytotoxicity=100%×[1−(LUCsample−LUCmin)/(LUCmax−LUCmin)]. The luciferase value ("LUC" or luminescence) is proportional to the amount of viable cells in each reaction well.

As shown in Figure 3A, LIC2001-1 expressing T cells (76.63 ± 4.58%), LIC2002-2 expressing T cells (96.52 ± 1.68%), and LIC2004 expressing T cells (96.89 ±0.70%) showed a significant killing effect against K562-CD123-Luc. As shown in Figure 3B, the killing effect was also observed on K562-Luc cells: LIC2001-1 expressing T cells (35.98 ± 6.08%), LIC2002-2 expressing T cells (94.42%) ±2.66%), LIC2004-expressing T cells (95.87±1.61%). However, engineered T cells expressing various NKG2DxIL-3 chimeric receptor constructs showed higher cytotoxicity towards K562-CD123-Luc cells than K562-Luc cells.

LIC2002-2 expressing T cells and LIC2004 expressing T cells at a ratio of effector (engineered T cells) to target cells of 10:1, 5:1, or 2.5:1 in human acute myeloid leukemia (AML). The cells were co-incubated with the cell line KG1-Luc and the in vitro cytotoxicity against KG1-Luc was evaluated. As shown in Figure 3C, LIC2002-2 expressing T cells (83.68 ± 7%, 78.55 ± 4%, and 60.87 ± 10%) and LIC2004 expressing T cells (85.6 ± 3%, 76 .89±4%, and 75.53±1%), a strong, dose-dependent cytotoxic effect was observed.

The cytotoxic activity of LIC2002-2-expressing T, LIC2004-expressing T, and LIC2004-1-expressing T against K562-CD123-Luc was compared at various effector:target ratios. The LIC2004-1 construct is the NKG2D chimeric receptor in the LIC2004 dual chimeric receptor system. The results are shown in Figure 4. The Y-axis shows the percentage of specific killing and the X-axis shows the natural logarithm of the effector:target ratio (ie, engineered T cells:K562-CD123-Luc cells). The logarithm of the E:T ratio is plotted against the percentage of specific kill and is fitted to the dotted line by linear regression. The smaller the slope of the fitted line, the stronger the killing ability. The results showed that the LIC2004 dual chimeric receptor system had higher efficacy than the corresponding NKG2D chimeric receptor alone (ie, LIC2004-1) (p=0.031). There is no significant difference between the efficacy of bispecific chimeric receptor constructs LIC2002-2 and LIC2004 (p=0.277). Statistical analysis was performed using Graphpad Prism6.
Mechanism of action

To investigate the mechanism of engineered T cells expressing the NKG2DxIL-3 chimeric receptor construct, a series of in vitro cytotoxicity assays were performed. First, "NKG2D-CD123T cells" (LIC2004-expressing T cells), "NKG2D T cells" (T cells expressing NKG2D-CD8hinge-CD8TM-4-1BB-CD3ζ), and "CD123 binder T cells" ( T cells expressing IL3-CD8hinge-CD8TM) were co-cultured with target K562-CD123-luc cells and K562-luc cells at an E:T ratio of 20:1, respectively.

As shown in Figure 5A, NKG2D-CD123 T cells showed a significant tumor killing effect (70.59 ± 1.5%), while the tumor killing effect of NKG2D T cells (42.14 ± 8.4%) was much weaker, and CD123 binder T cells showed no cytotoxicity against target tumor cells. In Figure 5B, NKG2D-CD123 T cells also showed significant cytotoxicity (88.28 ± 6.58%) against K562-luc cells, while NKG2D T cells exhibited lower cytotoxicity (66 CD123 binder T cells had no cytotoxicity against K562-luc cells (-46.98±11.22%). These results indicate that NKG2D-CD123 T cells are more effective than NKG2D T cells.

Additionally, cytotoxicity assays were performed in the presence or absence of NKG2D's cognate ligand, soluble MICA. When NKG2D-CD123 T cells (LIC2002-2 or LIC2004) were co-cultured with target cells (K562-Luc or K562-CD123-Luc) at an E:T ratio of 20:1, recombinant MICA protein or BSA protein Blocked each. MICA or BSA was added to the co-cultures at concentrations of 0 ng/mL, 100 ng/mL, or 1000 ng/mL. T cells electroporated without mRNA served as a negative control.

As shown in FIGS. 6A-6B, upon treatment with the highest concentration of MICA tested, the NKG2D domain could be blocked and the cytotoxic activity of LIC2002-2 and LIC2004-expressing T cells could be reduced. Treatment with non-specific BSA did not significantly affect the cytotoxicity of engineered T cells towards tumor cells.

As shown in Figure 7, LIC2002-2 expressing T cells and LIC2004 expressing T cells were combined at ratios of effector (engineered T cells) to target cells of 20:1, 10:1, 5:1, 2.5:1. , 1.25:1, or 0.625:1 with K562-CD123-Luc or K562-Luc cell lines. Results show dose-dependent killing effects of LIC2004 and LIC2002-2 on K562 cell line. A stronger killing effect was observed against the K562 cell line expressing both NKG2D and CD123 than against the K562 cell line expressing NKG2D alone.
IFNγ release

Engineered T cells expressing LIC2002-2, LIC2004, or LIC2004-1 constructs were co-incubated with K562-CD123-Luc, K562-Luc, or KG1-Luc cell lines for 20 hours, respectively. Supernatants from co-cultures were collected and evaluated to determine the level of cytokine release (eg, interferon gamma, IFNγ release).

Figure 8A shows the levels of IFNγ in cell-free supernatants after co-culturing engineered T cells with CD123-positive K562-CD123-Luc for 20 hours. The levels of secreted IFNγ were 1526.51 ± 92.13 pg/mL (LIC2002-2), 1089.36 ± 8.06 pg/mL (LIC2004), and 687.62 ± 31.65 pg/mL (LIC2004-1). , and 64.15±16.56 pg/mL (no RNA control). IFNγ was not detectable in the no T cell controls.

Figure 8B shows the levels of IFNγ in the cell-free supernatant after co-culturing engineered T cells with K562-Luc for 20 hours. The levels of secreted IFNγ were 3416.67 ± 71.15 pg/mL (LIC2002-2), 3063.46 ± 119.46 pg/mL (LIC2004), and 1841.41 ± 222.18 pg/mL (LIC2004-1). , and 3.99±11.57 pg/mL (no RNA control). IFNγ was not detectable in the no T cell controls.

Figure 8C shows the levels of IFNγ in the cell-free supernatant after co-culturing engineered T cells with KG1-Luc for 20 hours. The levels of secreted IFNγ were 267.75 ± 34.33 pg/mL (LIC2002-2), 265.87 ± 12.19 pg/mL (LIC2004), and 236.56 ± 16.45 pg/mL (LIC2004-1). , and 220.56±80.5 pg/mL (no RNA control). IFNγ was not detectable in the no T cell controls.
[Item 1]
A chimeric receptor having a polypeptide chain, the polypeptide chain comprising:
(a) an extracellular domain containing an NKG2D domain;
(b) a transmembrane domain;
(c) an intracellular signaling domain;
including,
Chimeric receptor.
[Item 2]
A multispecific chimeric receptor having a polypeptide chain, the polypeptide chain comprising:
(a) an extracellular domain comprising a first NKG2D domain, a second NKG2D domain, and a second antigen-binding domain;
(b) a transmembrane domain;
(c) an intracellular signaling domain;
including,
Multispecific chimeric receptors.
[Item 3]
The multispecific chimeric receptor according to item 2, wherein the extracellular domain comprises, from N-terminus to C-terminus, a second antigen-binding domain, a first NKG2D domain, and a second NKG2D domain.
[Item 4]
Chimeric receptor according to any one of items 1 to 3, wherein the second antigen binding domain is fused to the first NKG2D domain via a peptide linker.
[Item 5]
A multispecific chimeric receptor having a first polypeptide chain and a second polypeptide chain, each polypeptide chain comprising:
(a) an extracellular domain comprising an NKG2D domain and a second antigen binding domain;
(b) a transmembrane domain;
(c) an intracellular signaling domain;
Multispecific chimeric receptors.
[Item 6]
Multispecific chimeric receptor according to item 5, wherein each extracellular domain further comprises a dimerization motif.
[Item 7]
Multispecific chimeric receptor according to item 6, wherein the dimerization motif is located between the NKG2D domain and the second antigen binding domain.
[Item 8]
Multispecific chimeric receptor according to item 6 or 7, wherein the dimerization motif is a leucine zipper.
[Item 9]
the second antigen binding domain is fused to the NKG2D domain via a peptide linker;
Multispecific chimeric receptor according to item 8.
[Item 10]
Each of the first polypeptide chain and the second polypeptide chain comprises, from the N-terminus to the C-terminus, a second antigen-binding domain, an NKG2D domain, a transmembrane domain, and an intracellular signaling domain.
Multispecific chimeric receptor according to any one of items 5 to 9.
[Item 11]
A dual chimeric receptor system,
(i) a first chimeric receptor,
(a) a first extracellular domain comprising an NKG2D domain;
(b) a first transmembrane domain;
(c) a first intracellular signaling domain;
receptor and
(ii) a second chimeric receptor,
(a) a second extracellular domain comprising a second antigen-binding domain;
(b) having a second transmembrane domain;
receptor and
Equipped with
Dual chimeric receptor system.
[Item 12]
12. The dual chimeric receptor system of item 11, wherein the second chimeric receptor further comprises a second intracellular signaling domain.
[Item 13]
The multispecific chimeric receptor according to any one of items 2 to 10, or the dual chimeric receptor system according to items 11 or 12, wherein the second antigen binding domain is an antibody fragment.
[Item 14]
Antibody fragments include CD19, CD20, CD22, CD33, CD38, BCMA, CS1, ROR1, GPC3, CD123, CD138, c-Met, EGFR, EGFRvIII, HER2, HER3, GD-2, NY-ESO-1, MAGE A3 14. The multispecific chimeric receptor or dual chimeric receptor system according to item 13, which specifically binds to an antigen selected from the group consisting of , and glycolipid F77.
[Item 15]
A multispecific chimeric receptor according to any one of items 2 to 10, or a dual chimeric receptor system according to items 11 or 12, wherein the second antigen binding domain is a ligand or a ligand binding domain.
[Item 16]
The multispecific compound according to item 15, wherein the ligand or ligand binding domain is derived from a molecule selected from the group consisting of NKG2A, NKG2C, NKG2F, IL-3, IL-13, LLT1, AICL, DNAM-1, and NKp80. Sexual chimeric receptors or dual chimeric receptor systems.
[Item 17]
Multispecific chimeric receptor or dual chimeric receptor system according to item 16, wherein the second antigen binding domain is an IL-3 domain.
[Item 18]
The NKG2D domain (or the first NKG2D domain and/or the second NKG2D domain) comprises the multispecificity according to any one of items 1 to 10 and 13 to 17, wherein the NKG2D domain (or the first NKG2D domain and/or the second NKG2D domain) comprises the amino acid sequence of SEQ ID NO: 7 or 8. A chimeric receptor or a dual chimeric receptor system according to any one of items 11 to 17.
[Item 19]
The transmembrane domain (or the first transmembrane domain and/or the second transmembrane domain) is a molecule selected from the group consisting of CD8α, CD4, CD28, 4-1BB, CD80, CD86, CD152, and PD1. A chimeric receptor according to any one of items 1 to 10 and 13 to 18, or a dual chimeric receptor system according to any one of items 11 to 18, derived from the invention.
[Item 20]
Items 1-10 and 13-19, wherein the intracellular signaling domain (or first intracellular signaling domain and/or second intracellular signaling domain) comprises a primary intracellular signaling domain of an immune effector cell. or the dual chimeric receptor system according to any one of items 11 to 19.
[Item 21]
The intracellular signaling domain (or the first intracellular signaling domain and/or the second intracellular signaling domain) comprises any one of items 1 to 10 and 13 to 20, comprising a costimulatory signaling domain. or the dual chimeric receptor system according to any one of items 11 to 20.
[Item 22]
Co-stimulatory signaling domains include ligands for CD27, CD28, 4-1BB, OX40, CD30, ICOS, CD40, CD3, LFA-1, CD2, CD7, LIGHT, NKG2C, B7-H3, CD83, and combinations thereof. 22. The chimeric receptor or dual chimeric receptor system according to item 21, derived from a costimulatory molecule selected from the group consisting of:
[Item 23]
A chimeric receptor comprising an amino acid sequence having at least about 85% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 16-20 and 33-35.
[Item 24]
A polypeptide comprising an amino acid sequence having at least about 85% sequence identity to the amino acid sequence of SEQ ID NO: 36 or 37.
[Item 25]
An isolated nucleic acid comprising a nucleic acid sequence encoding a chimeric receptor according to any one of items 1-10 and 13-23.
[Item 26]
The double chimeric receptor according to any one of items 11 to 22, comprising a first nucleic acid sequence encoding a first chimeric receptor and a second nucleic acid sequence encoding a second chimeric receptor. An isolated nucleic acid encoding a system, wherein a first nucleic acid sequence is operably linked to a second nucleic acid sequence via a third nucleic acid sequence encoding a self-cleavable peptide.
[Item 27]
An isolated nucleic acid comprising a nucleic acid sequence having at least about 85% sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 21-27 and SEQ ID NOs: 38-40.
[Item 28]
A modified immune effector cell comprising a chimeric receptor or dual chimeric receptor system according to any one of items 1 to 23, or an isolated nucleic acid according to any one of items 25 to 27.
[Item 29]
A pharmaceutical composition comprising the modified immune effector cell according to item 28 and a pharmaceutically acceptable carrier.
[Item 30]
A method of treating cancer in an individual, comprising administering to the individual an effective amount of the pharmaceutical composition according to item 29.

Claims (18)

A multispecific chimeric receptor having a polypeptide chain, the polypeptide chain comprising:
(a) an extracellular domain comprising a first NKG2D domain, a second NKG2D domain, and a second antigen-binding domain;
(b) a transmembrane domain;
(c) an intracellular signaling domain;
including;
The first NKG2D domain comprises the amino acid sequence of SEQ ID NO: 7 ,
The second NKG2D domain comprises the amino acid sequence of SEQ ID NO: 8 ,
the first NKG2D domain and the second NKG2D domain form a dimer;
Multispecific chimeric receptors. The multispecific chimeric receptor of claim 1, wherein the extracellular domain comprises, from N-terminus to C-terminus, the second antigen-binding domain, the first NKG2D domain, and the second NKG2D domain. . Multispecific chimeric receptor according to claim 1 or 2, wherein the second antigen binding domain is fused to the first NKG2D domain via a peptide linker. Multispecific chimeric receptor according to any one of claims 1 to 3 , wherein the second antigen binding domain is an antibody fragment. The antibody fragments include CD19, CD20, CD22, CD33, CD38, BCMA, CS1, ROR1, GPC3, CD123, CD138, c-Met, EGFR, EGFRvIII, HER2, HER3, GD-2, NY-ESO-1, MAGE. 5. The multispecific chimeric receptor of claim 4 , which specifically binds to an antigen selected from the group consisting of A3 and glycolipid F77. Multispecific chimeric receptor according to any one of claims 1 to 3 , wherein the second antigen binding domain is a ligand or a ligand binding domain. 7. The ligand or ligand binding domain is derived from a molecule selected from the group consisting of NKG2A, NKG2C, NKG2F, IL-3, IL-13, LLT1, AICL, DNAM-1, and NKp80. Multispecific chimeric receptors. 8. The multispecific chimeric receptor of claim 7 , wherein the second antigen binding domain is an IL-3 domain. 9. The multiplex according to any one of claims 1 to 8 , wherein the transmembrane domain is derived from a molecule selected from the group consisting of CD8α, CD4, CD28, 4-1BB, CD80, CD86, CD152, and PD1. Specificity chimeric receptor. 10. The multispecific chimeric receptor of any one of claims 1 to 9 , wherein the intracellular signaling domain comprises a primary intracellular signaling domain of an immune effector cell. 11. The multispecific chimeric receptor of any one of claims 1 to 10 , wherein the intracellular signaling domain comprises a costimulatory signaling domain. The costimulatory signaling domain includes ligands for CD27, CD28, 4-1BB, OX40, CD30, ICOS, CD40, CD3, LFA-1, CD2, CD7, LIGHT, NKG2C, B7-H3, CD83, and combinations thereof. Multispecific chimeric receptor according to claim 11 , derived from a costimulatory molecule selected from the group consisting of. Multispecific chimeric receptor according to any one of claims 1 to 12 , comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 16 and 17. An isolated nucleic acid comprising a nucleic acid sequence encoding a multispecific chimeric receptor according to any one of claims 1 to 13 . An isolated nucleic acid comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 21 and 22. A modified immune effector cell comprising a multispecific chimeric receptor according to any one of claims 1 to 13 or an isolated nucleic acid according to claims 14 or 15 . A pharmaceutical composition comprising a modified immune effector cell according to claim 16 and a pharmaceutically acceptable carrier. 18. A pharmaceutical composition according to claim 17 for use in the treatment of cancer.